Non-reciprocal recombination-mediated transgene deletion in transgenic plants

ABSTRACT

The invention provides a process to prepare a recombined transgenic  Zea mays  plant from a transgenic  Zea mays  plant, wherein the transgene in the recombinant plant has an altered genetic structure relative to the genetic structure of the transgene in the transgenic plant, due to recombination-mediated transgene deletion.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for producing transgenicplants, particularly transgenic plants from which ancillary sequences,such as vector DNA or reporter or selection genes have been deleted.More specifically, the invention relates to a method for deletingancillary sequences derived from gene transfer vectors frommonocotyledonous transgenic plants.

2. Description of the Related Art

Genetically modified (GM) crops offer many advantages to the farmer interms of inputs to crop production, e.g. weed and insect control, andimproved usage of water and nutrient inputs. Genetically modified plantsalso provide a means for improving nutritional value, e.g. improvedamino acid or protein composition, improved starch and oil quantitiesand qualities, increased vitamin levels, or bioavailability ofnutrients, or can be the source of pharmaceuticals or “nutraceuticals”.Methods have been developed for conferring tolerance or resistance towater or salt stress in monocots (U.S. Pat. No. 5,780,709), for example,and a single gene has been used to improve tolerance to drought, saltloading, and freezing in some plants. (Kasuga et al., 1999). Insectresistance can be conferred to transferring genes for the production oftoxins found in the soil bacterium Bacillus thuringiensis (Bt). Lysinecontent has been increased by incorporating the genes for bacterialenzymes (e.g. Corynebacterium dihydropicolinic acid synthase and E. coliaspartokinase) into GM plants. The comparable plant enzymes are subjectto lysine feedback inhibition, while the bacterial enzymes show littleor no feedback inhibition.

Until technology made genetic modification of plants possible,production of plants with desirable characteristics was dependent uponselective breeding and the variability naturally present in a crop andclosely related sexually compatible species. Genetic modificationthrough transformation provides an efficient and controlled method forproducing plants with one or more desired characteristics, includingcharacteristics that are normally not found in those crops, such asresistance to herbicides or pests, or nutritionally balanced food orfeed products.

Genetic modification of crops by transformation sometimes involvestransfer of one or more desired genes, along with ancillary sequencessuch as antibiotic resistance markers or reporter genes, into a plantcell. Antibiotic resistance markers used in plant genetic engineering,for example, include the kanamycin resistance marker (Carrer et al.,1993), streptomycin resistance marker (Moll et al., 1990), lincomycinresistance marker (Jenkins et al., 1991) and the neomycin resistancemarker (Beck et al., 1982). The ancillary sequences are necessary foridentification or selection of transformed cells, but do not contributeto the trait conferred on the plant. Since ancillary sequences do notcontribute to the desired crop improvement, efforts have been made todelete them from the GM progeny. Antibiotic resistance markers haveparticularly been targeted for deletion.

Furthermore, it has been demonstrated that using direct DNA deliverymethods, such as microprojectile bombardment, complex transgeneinsertions may occur including duplications, deletions, and complexrearrangements of introduced DNA (PCT Publication No. WO 99/32642).Complex transgene insertions may contribute to co-suppression of geneexpression or genetic instability of the insertion.

A number of site-specific recombination-mediated methods have beendeveloped for incorporating target genes into plant genomes, as well asfor deleting unwanted genetic elements from plant and animal cells. Forexample, the cre-lox recombination system of bacteriophage P1, describedby Abremski et al. (1983), Sternberg el al. (1981) and others, has beenused to promote recombination in a variety of cell types. The cre-loxsystem utilizes the cre recombinase isolated from bacteriophage P1 inconjunction with the DNA sequences (termed lox sites) it recognizes.This recombination system has been effective for achieving recombinationin plant cells (U.S. Pat. No. 5,658,772), animal cells (U.S. Pat. No.4,959,317 and U.S. Pat. No. 5,801,030), and in viral vectors (Hardy etal., 1997).

Wahl et al. (U.S. Pat. No. 5,654,182) used the site-specific FLPrecombinase system of Saccharomyces cerevisiae to delete DNA sequencesin eukaryotic cells. The deletions were designed to accomplish eitherinactivation of a gene or activation of a gene by bringing desired DNAfragments into association with one another.

Others have used transposons, or mobile genetic elements that transposewhen a transposase gene is present in the same genome, to separatetarget genes from ancillary sequences. Yoder el al. (U.S. Pat. No.5,482,852 and U.S. Pat. No. 5,792,924) used constructs containing thesequence of the transposase enzyme and the transposase recognitionsequences to provide a method for genetically altering plants thatcontain a desired gene free of vector and/or marker sequences.

Oliver et al. (U.S. Pat. No. 5,723,765) used site-specific recombinationsystems in conjunction with a blocking sequence to provide a regulatorymechanism in transgenic plants. In this method, when site-specificrecombination results in excision of the blocking sequence, regulatoryelements that either induce or repress a particular gene of interest aremoved into an appropriate position upstream from the target sequence.

Although each of these methods has been designed specifically to exciseunwanted sequences, each also relies upon introduction of ancillarygenetic sequences (e.g., recombinase or transposase specific recognitionsequences) that ultimately do not contribute to the desired cropimprovement.

Thus, there is a need for a method for excising unwanted DNA sequencesfrom transgenic plants without introducing any further ancillary DNAsequences.

SUMMARY OF THE INVENTION

The invention provides a novel method for excision, modification, oramplification of DNA sequences from transgenic plants that does notinvolve the use of site-specific recombination enzymes, includingtransposable elements, but instead relies upon homologous sequencespositioned about the target sequence to direct excision or amplificationthrough native cellular recombination mechanisms.

The invention provides a novel method of removing undesirable DNAsequences as well as a method for resolving complex transgene insertionsto simpler insertions, thereby increasing transgene stability anddecreasing the occurrence of co-suppression.

The invention provides a method of preparing a recombined fertiletransgenic plant, by obtaining a first fertile transgenic plant having afirst DNA sequence, a second DNA sequence operably linked to at leastone regulatory sequence functional in a plant cell, and a third DNAsequence homologous to at least a portion of the first DNA sequence andpositioned so that the directly repeated DNA sequences flank the secondDNA sequence. Additionally, the transgene insertion may comprise furtherDNA sequences. In the method of the present invention, the direct repeatmay be recognized by a site-specific recombinase enzyme, but a sitespecific recombinase is not required for deletion of the desiredsequence. The first fertile transgenic plants are crossed to produceeither hybrid or inbred progeny plants, and from those progeny plantsone or more second fertile transgenic plants are selected which containa second DNA sequence that has been altered by recombination. The firstfertile transgenic plant can be either homozygous or hemizygous for thesecond DNA sequence.

The invention provides a method of preparing a fertile transgenic planthaving an altered transgene insertion comprising obtaining a firstfertile transgenic plant homozygous for a transgene insertion DNAsequence, wherein the transgene insertion DNA sequence comprises apre-selected DNA sequence flanked by directly repeated DNA sequences,obtaining a plurality of progeny of any generation of the firsttransgenic plant, and selecting a progeny fertile transgenic plantwherein the transgene insertion is altered as compared to the firstfertile transgenic plant. Methods are provided wherein the pre-selectedDNA sequence comprises a selectable marker gene or a reporter gene, suchas a bar, nptII or cryIA(b) gene. Furthermore, methods are providedwherein the plurality of progeny plants are obtained by eitherself-pollination or outcrossing. The resultant progeny plants may beeither inbreds or hybrids. The plants may be a cereal plant, such as amaize, barley, wheat rye or rice plant.

Also provided by the present invention is a transgenic plant produced bythe method, wherein the transgene insertion is altered as compared tothe first fertile transgenic plant.

The invention also provides a seed for producing the recombinanttransgenic plant, wherein the transgene insertion is altered as comparedto the first fertile transgenic plant.

Also provided is a fertile transgenic plant wherein a transgeneinsertion is altered from a parent transgene insertion. The plant may behybrid or inbred. The transgene insertion may be altered in that it hasbeen deleted, amplified, or rearranged.

Further provided is a progeny plant of any generation comprising analtered transgene insertion, wherein the transgene insertion is alteredfrom the transgene insertion in a parental R₀ plant.

The present invention also provides an altered transgene insertion DNAsequence preparable by the method comprising obtaining a first fertiletransgenic plant homozygous for a transgene insertion DNA sequence,wherein the transgene DNA sequence comprises a pre-selected DNA sequenceflanked by directly repeated DNA sequences; obtaining a plurality ofprogeny of any generation of the first transgenic plant; and selecting aprogeny fertile transgenic plant wherein the transgene insertion isaltered as compared to the first fertile transgenic plant. The transgeneinsertion may be altered in that it has been deleted, amplified, orrearranged. Alteration of the transgene insertion may result in a changein expression of a transgene contained within the parental transgeneinsertion. The alteration of the transgene may be identified by DNAanalysis, such as by PCR or Southern blot analysis. The alteredtransgene insertion may be in a cereal plant, such as a maize, barley,wheat, rye or rice plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Direct repeat induced non-reciprocal recombination-mediatedalteration of a transgene insertion.

FIG. 2. DBT418 transgene insertion indicating direct repeat sequencesthat were the substrate for non-homologous recombination productsrecovered in the 09-07 and 03-09 altered transgene insertion progeny.

FIG. 3. Altered transgene insertions recovered from DBT418.

FIG. 4. MON849 transgene insertion and altered insertions recoveredfollowing non-homologous recombination.

FIG. 5. Plasmid vector pDPG354.

FIG. 6. Plasmid vector pMON19344.

FIG. 7. Plasmid vector pDPG165.

FIG. 8. Plasmid vector pDPG320.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a novel method for production of transgenicplants lacking ancillary DNA sequences that do not contribute to thedesired phenotypic trait. The inventors have discovered that homologousrecombination occurs in plants at a rate sufficient to providetransgenic progeny without added recombinase enzymes. In the methodprovided, homologous sequences are located 5′, 3′, or both 5′ and 3′ toa target sequence, to be amplified within, modified, or excised from theplant genome. The inventors have determined that gene deletionfrequency, for example, is approximately 0.1% per 287±19 base pairs ofhomologous direct repeat sequence. The method described herein can beused to delete selectable marker genes, to delete partial or rearrangedgene copies, to reduce overall transgene copy number, or to increaseoverall transgene copy number.

In non-reciprocal homologous recombination, which can be usedparticularly to produce transgene deletions, direct repeats that arepresent in the introduced DNA sequence, or produce a DNA alignment,result in amplification of the number of copies of a particular genesequence or excision of either one or more ancillary DNA sequences ortarget DNA sequences. This method can be used to delete ancillarysequences or to remove regulatory sequences, for example, in order toactivate or deactivate a target gene.

For some time, it has been known that homologous recombination resultsin genetic rearrangements. What has not been known, however, is thathomologous recombination, which is not dependent upon added recombinaseenzymes, can be used in plant cells to amplify DNA sequences or toexcise target sequences. In the method of the present invention,non-reciprocal homologous recombination (which has sometimes been knownas gene conversion), provides non-reciprocal transfer of geneticinformation between two related DNA sequences, often resulting inaddition of genetic information to one allele with associated deletionof genetic information from the corresponding allele.

Non-reciprocal homologous recombination is illustrated generally in FIG.1. In homologous recombination, similar alleles contain similar DNAsequences at their 5′ and 3′ ends. These are often referred to ashomologous sequences. If these sequences are located at a sufficientdistance apart, base-pairing resulting from the homology can occurduring meiosis. This results in “crossing-over” that leads to arecombination event, exchanging the genetic information of one allelefor the genetic information of a second allele, as shown in FIG. 1. Innon-reciprocal homologous recombination, however, regions of homologyare positioned so as to generally result in an uneven (usually one)number of cross-over events, leading to an uneven exchange of geneticmaterial, as illustrated in FIG. 1. An uneven cross-over event usuallyresults in addition of genetic information to one allele at the expenseof a deletion of genetic information from its corresponding allele, asillustrated in FIG. 1. By producing a transgene construct thatincorporates DNA sequence homologies at desired locations, it ispossible to enhance the frequency of such uneven cross-over events intransgenic plant cells, resulting in targeted deletion or amplificationof desired DNA sequences in progeny cells.

The method of the present invention can be used with a variety ofplants, and is especially useful for development of transgenic cerealplants, such as maize, barley, wheat, rye or rice.

II. Definitions

The following words and phrases have the meanings set forth below.

Chimeric gene: A gene or DNA sequence or segment comprising at least twoDNA sequences or segments from species that do not combine DNA undernatural conditions, or DNA sequences or segments that are positioned orlinked in a manner that does not normally occur in the native genome ofan untransformed plant, such as maize or another monocot.

Exogenous gene: A gene that is not normally present in a given hostgenome in the exogenous gene's present form. In this respect, the geneitself may be native to the host genome, however the exogenous gene willcomprise the native gene altered by the addition or deletion of one ormore different regulatory elements.

Expression: The combination of intracellular processes, includingtranscription and translation undergone by a coding DNA molecule such asa structural gene to produce a polypeptide.

Expression cassette: A nucleic acid segment comprising at least a firstgene that one desires to have expressed in a host cell and the necessaryregulatory elements for expressing the gene in the host cell. Preferredregulatory elements for use with the invention include promoters,enhancers and terminators. It also may be desirable to include on theexpression cassette a nucleic acid segment encoding an appropriatetransit peptide, as is described below. The expression cassette may becontained and propagated in any suitable cloning vector, for example, aplasmid, cosmid, bacterial artificial chromosome, or yeast artificialchromosome. The whole vector DNA may be used to transform a host cell,or alternatively, the expression cassette may be isolated from thevector and then used for transformation.

Expression vector: A vector comprising at least one expression cassette.

Heterologous DNA: DNA from a source different from that of the recipientcell.

Homologous DNA: DNA from the same source as that of the recipient cell.

Obtaining: When used in conjunction with a transgenic plant cell ortransgenic plant, obtaining means either transforming a non-transgenicplant cell or plant to create the transgenic plant cell or plant, orplanting transgenic plant seed to produce the transgenic plant cell orplant.

Progeny: Any subsequent generation, including the seeds and plantstherefrom, which is derived from a particular parental plant or set ofparental plants.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provide an expression control element for a structural gene and towhich RNA polymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

R₀ Transgenic Plant: A plant that has been directly transformed withselected DNA or has been regenerated from a cell or cell cluster thathas been transformed with a selected DNA.

Recombined transgenic: A transgenic plant cell, plant part, plant tissueor plant, the transgenic DNA sequences or genes of which are altered bynon-reciprocal homologous recombination. Altered includes deleted,amplified, or any other modification of the preselected DNA sequence asoriginally integrated into the host genome.

Regeneration: The process of growing a plant from a plant cell (e.g.,plant protoplast, callus or explant).

Selected DNA: A DNA segment that one desires to introduce into a plantgenome by genetic transformation.

Selected gene: A gene that one desires to have expressed in atransformed plant, plant cell or plant part. A selected gene may benative or foreign to a host genome, but where the selected gene isnative to the host genome, will include one or more regulatory orfunctional elements that differ from native copies of the gene.

Structural gene: A gene that is expressed to produce a polypeptide.

Tranformation: A process of introducing an exegenous DNA sequence (e.g.,a vector, a recombinant DNA molecule) into a cell or protoplast in whichthat exogenous DNA is incorporated into a chromosome or is capable ofautonomous replication.

Transformation construct: A chimeric DNA molecule that is designed forintroduction into a host genome by genetic transformation. Preferredtransformation constructs will comprise all of the genetic elementsnecessary to direct the expression of one or more exogenous genes. Inparticular embodiments of the instant invention, it may be desirable tointroduce a transformation construct into a host cell in the form of anexpression cassette.

Transformed cell: A cell wherein its DNA has been altered by theintroduction of an exogenous DNA molecule into that cell.

Transgene: A segment of DNA that has been incorporated into a hostgenome or is capable of autonomous replication in a host cell and iscapable of causing the expression of one or more cellular products.Exemplary transgenes will provide the host cell, or plants regeneratedtherefrom, with a novel phenotype relative to the correspondingnon-transformed cell or plant. Transgenes may be directly introducedinto a plant by genetic transformation, or may be inherited from a plantof any previous generation that was transformed with the DNA segment.

Transgene insertion: A segment of DNA incorporated into a host genome. Atransgene insertion comprises all of the DNA sequences that wereintroduced by transformation and are present at a single genetic locusin a transformed cell or plant. DNA sequences within the transgeneinsertion may arise from one or more plasmid vectors. Furthermore, DNAsequences may be rearranged in a transgene insertion when compared tothe arrangement of DNA sequences in the parent plasmid vector orvectors. A transgene insertion may be altered using the methods of thisinvention, resulting in deletion, duplication, or rearrangement of DNAsequences. A parent transgene insertion is the original transgeneinsertion in a parent plant. The parent transgene insertion may bealtered by non-reciprocal recombination during a cycle of meiosis andthen transmitted to the progeny as an altered transgene insertion.

Transgenic cell: Any cell derived or regenerated from a transformed cellor derived from a transgenic cell. Exemplary transgenic cells includeplant calli derived from a transformed plant cell and particular somaticcells such as leaf, root, stem, or reproductive (germ) cells obtainedfrom a transgenic plant.

Transit peptide: A polypeptide sequence that is capable of directing apolypeptide to a particular organelle or other location within a cell.

Vector: A DNA molecule capable of replication in a host cell and/or towhich another DNA segment can be operatively linked so as to bring aboutreplication of the attached segment. A plasmid is an exemplary vector.

Wild type: An untransformed plant cell, plant part, plant tissue orplant wherein the genome has not been altered by the presence of apreselected DNA sequence.

II. DNA Constructs of the Invention

Virtually any DNA may be used for delivery to recipient cells toultimately produce fertile transgenic plants in accordance with thepresent invention. For example, an isolated and purified DNA segment inthe form of vectors and plasmids encoding a desired gene product orlinear DNA fragments, in some instances containing only the DNA elementto be expressed in the plant, and the like, may be employed.

DNA useful for introduction into plant cells includes that which hasbeen derived or isolated from any source, that may be subsequentlycharacterized as to structure, size and/or function, chemically altered,and later introduced into a plant. An example of DNA “derived” from asource, would be a DNA sequence or segment that is identified as auseful fragment within a given organism, and is then chemicallysynthesized in essentially pure form. An example of such DNA “isolated”from a source would be a useful DNA sequence that is excised or removedfrom said source by chemical means, e.g., by the use of restrictionendonucleases, so that it can be further manipulated, e.g., amplified,for use in the invention, by the methodology of genetic engineering.Such DNA is commonly referred to as “recombinant DNA.”

Therefore, useful DNA includes completely synthetic DNA, semi-syntheticDNA, DNA isolated from biological sources, and DNA derived from RNA. Itis within the scope of the invention to isolate and purify a DNA segmentfrom a given genotype, and to subsequently introduce multiple copies ofthe isolated and purified DNA segment into the same genotype, e.g., toenhance production of a given gene product.

The introduced DNA includes, but is not limited to, DNA from plant genesand non-plant genes, such as those from bacteria, yeasts, animals orviruses. The introduced DNA can include modified genes, portions ofgenes, or chimeric genes, including genes from the same or differentgenotype.

An isolated and purified DNA segment, molecule or sequence can beidentified and isolated by standard methods, as described by Sambrook etal. (1989). The isolated and purified DNA segment can be identified byscreening of a DNA or cDNA library generated from nucleic acid derivedfrom a particular cell type, cell line, primary cells, or tissue.Screening for DNA fragments that encode all or a portion of the isolatedand purified DNA segment can be accomplished by screening plaques from agenomic or cDNA library for hybridization to a probe of the DNA fromother organisms or by screening plaques from a cDNA expression libraryfor binding to antibodies that specifically recognize the proteinencoded by the isolated and purified DNA segment. DNA fragments thathybridize to an isolated and purified DNA segment probe from otherorganisms and/or plaques carrying DNA fragments that are immunoreactivewith antibodies to the protein encoded by the isolated and purified DNAsegment can be subcloned into a vector and sequenced and/or used asprobes to identify other cDNA or genomic sequences encoding all or aportion of the isolated and purified DNA segment.

Portions of the genomic copy or copies of the isolated and purified DNAsegment can be sequenced and the 5′ end of the DNA identified bystandard methods including either DNA sequence homology to otherhomologous genes or by RNAase protection analysis, as described bySambrook et al. (1989). Once portions of the 5′ end of the isolated andpurified DNA segment are identified, complete copies of the isolated andpurified DNA segment can be obtained by standard methods, includingcloning or polymerase chain reaction (PCR) synthesis usingoligonucleotide primers complementary to the isolated and purified DNAsegment at the 5′ end of the DNA. The presence of an isolatedfull-length copy of the isolated and purified DNA can be verified byhybridization, partial sequence analysis, or by expression of theisolated and purified DNA segment.

The DNA may be circular or linear, double-stranded or single-stranded.Generally, the DNA is in the form of chimeric DNA that can also containcoding regions flanked by regulatory sequences that promote theexpression of the recombinant DNA present in the resultant plant (an“expression cassette”). For example, the DNA may itself comprise orconsist of a promoter that is active in which is derived from anon-source, or may utilize a promoter already present in the genotype.

Ultimately, the most desirable DNA segments for introduction into amonocot genome may be homologous genes or gene families that encode adesired trait (e.g., increased yield per acre) and that are introducedunder the control of novel promoters or enhancers, etc., or perhaps evenhomologous or tissue-specific (e.g., root-, collar/sheath-, whorl-,stalk-, earshank-, kernel- or leaf-specific) promoters or controlelements. Indeed, it is envisioned that a particular use of the presentinvention may be the targeting of an isolated and purified DNA segmentin a tissue- or organelle-specific manner.

The construction of vectors that may be employed in conjunction with thepresent invention will be known to those of skill of the art in light ofthe present disclosure (see, e.g., Sambrook et al., 1989; Gelvin et al.,1990).

Generally, the introduced DNA will be relatively small, i.e., less thanabout 30 kb to minimize any susceptibility to physical, chemical, orenzymatic degradation that is known to increase as the size of the DNAincreases. The number of proteins, RNA transcripts or mixtures thereofthat is introduced into the genome is preferably isolated and purifiedand defined, e.g., from one to about 5-10 such products of theintroduced DNA may be formed.

A. Expression Cassettes

1. Promoters, Enhancers and Other Non-3′ Transcription RegulatorySequences

Preferably, the expression cassette of the invention is operably linkedto a promoter, which provides for expression of a linked DNA sequence.The DNA sequence is operably linked to the promoter when it is locateddownstream from the promoter, to form an expression cassette. Anisolated promoter sequence that is a strong promoter for heterologousDNAs is advantageous because it provides for a sufficient level of geneexpression to allow for easy detection and selection of transformedcells and provides for a high level of gene expression when desired.

Most endogenous genes have regions of DNA that are known as promoters,which regulate gene expression. Promoter regions are typically found inthe flanking DNA upstream from the coding sequence in both prokaryoticand eukaryotic cells. A promoter sequence provides for regulation oftranscription of the downstream gene sequence and typically includesfrom about 50 to about 2,000 nucleotide base pairs. Promoter sequencesalso contain regulatory sequences such as enhancer sequences that caninfluence the level of gene expression. Some isolated promoter sequencescan provide for gene expression of heterologous DNAs, that is a DNAdifferent from the native or homologous DNA.

Promoter sequences are also known to be strong or weak, or inducible. Astrong promoter provides for a high level of gene expression, whereas aweak promoter provides for a very low level of gene expression. Aninducible promoter is a promoter that provides for the turning on andoff of gene expression in response to an exogenously added agent, or toan environmental or developmental stimulus. Promoters can also providefor tissue specific or developmental regulation.

Preferred expression cassettes will generally include, but are notlimited to, a plant promoter such as the CaMV 35S promoter (Odell etal., 1985), or others such as CaMV 19S (Lawton et al., 1987), nos (Ebertet al., 1987), Adh (Walker et al., 1987), sucrose synthase (Yang et al.,1990), α-tubulin, ubiquitin, actin (Wang et al., 1992), cab (Sullivan etal., 1989), PEPCase (Hudspeth et al., 1989) or those associated with theR gene complex (Chandler et al., 1989). Further suitable promotersinclude inducible promoters, such as the light inducible promoterderived from the pea rbcS gene (Coruzzi et al., 1971), the actinpromoter from rice (McElroy et al., 1990), and water-stress-, ABA- andturgor-inducible promoters (Skriver et al., 1990; Guerrero et al.,1990), tissue-specific promoters, such as root-cell promoters (Conklinget al., 1990), and developmentally-specific promoters such as seedspecific promoters, e.g., the phaseolin promoter from beans(Sengupta-Gopalan, 1985), and the Z10 and Z27 promoters from maize. Forexample, expression of zein storage proteins is initiated in theendosperm about 15 days after pollination. Tissue specific expressionmay also be functionally accomplished by introducing a constitutivelyexpressed gene (all tissues) in combination with an antisense gene thatis expressed only in those tissues where the gene product is notdesired.

Promoters that direct specific or enhanced expression in certain planttissues will be known to those of skill in the art in light of thepresent disclosure. These include, for example, the rbcS promoter,specific for green tissue; the ocs, nos and mas promoters that havehigher activity in roots or wounded leaf tissue; a truncated (−90 to +8)35S promoter that directs enhanced expression in roots, an α-tubulingene that directs expression in roots and promoters derived from zeinstorage protein genes that direct expression in endosperm. Transcriptionenhancers or duplications of enhancers can be used to increaseexpression from a particular promoter (see, for example, Fromm et al.,1989). Examples of such enhancers include, but are not limited to,elements from the CaMV 35S promoter and octopine synthase genes (U.S.Pat. No. 5,290,924). It is particularly contemplated that one mayadvantageously use the 16 bp ocs enhancer element from the octopinesynthase (ocs) gene (Ellis et al., 1987; Bouchez et al., 1989),especially when present in multiple copies, to achieve enhancedexpression in roots. Other promoters useful in the practice of theinvention are known to those of skill in the art. For example, see VanOoijen et al. (U.S. Pat. No. 5,593,963) and Walsh et al. (U.S. Pat. No.5,743,477).

Alternatively, novel tissue-specific promoter sequences may be employedin the practice of the present invention. cDNA clones from a particulartissue are isolated and those clones that are expressed specifically inthat tissue are identified, for example, using Northern blotting.Preferably, the gene isolated is not present in a high copy number, butis expressed in specific tissues. The promoter and control elements ofcorresponding genomic clones can then be localized using techniques wellknown to those of skill in the art.

As the DNA sequence inserted between the transcription initiation siteand the start of the coding sequence, i.e., the untranslated leadersequence, can influence gene expression, one can also employ aparticular leader sequence. Preferred leader sequence include those thatcomprise sequences selected to direct optimum expression of the attachedgene, i.e., to include a preferred consensus leader sequence that canincrease or maintain mRNA stability and prevent inappropriate initiationof translation (Joshi, 1987). Such sequences are known to those of skillin the art. Sequences that are derived from genes that are highlyexpressed in plants, and in in particular, will be most preferred.

Regulatory elements such as Adh intron 1 (Callis et al., 1987), sucrosesynthase intron (Vasil et al., 1989), rice actin 1 intron 1 (McElroy etal., 1991) or TMV omega element (Gallie et al., 1989) can also beincluded where desired. Other such regulatory elements useful in thepractice of the invention are known to those of skill in the art.

An isolated and purified DNA segment can be combined with thetranscription regulatory sequences by standard methods as described inSambrook et al., cited supra, to yield an expression cassette. Briefly,a plasmid containing a promoter such as the 35S CaMV promoter can beconstructed as described in Jefferson (1987) or obtained from ClontechLab in Palo Alto, Calif. (e.g., pBI121 or pBI221). Typically, theseplasmids are constructed to provide for multiple cloning sites havingspecificity for different restriction enzymes downstream from thepromoter. The isolated and purified DNA segment can be subcloneddownstream from the promoter using restriction enzymes to ensure thatthe DNA is inserted in proper orientation with respect to the promoterso that the DNA can be expressed. Once the isolated and purified DNAsegment is operably linked to a promoter, the expression cassette soformed can be subcloned into a plasmid or other vectors.

2. Targeting Sequences

Additionally, expression cassettes can be constructed and employed totarget the product of the isolated and purified DNA sequence or segmentto an intracellular compartment within plant cells or to direct aprotein to the extracellular environment. This can generally be achievedby joining a DNA sequence encoding a transit or signal peptide sequenceto the coding sequence of the isolated and purified DNA sequence. Theresultant transit, or signal, peptide will transport the protein to aparticular intracellular, or extracellular destination, respectively,and can then be post-translationally removed. Transit peptides act byfacilitating the transport of proteins through intracellular membranes,e.g., vacuole, vesicle, plastid and mitochondrial membranes, whereassignal peptides direct proteins through the extracellular membrane. Byfacilitating transport of the protein into compartments inside oroutside the cell, these sequences can increase the accumulation of aparticular gene product in a particular location. For example, see U.S.Pat. Nos. 5,258,300 and 5,593,963.

The isolated and purified DNA segment can be directed to a particularorganelle, such as the chloroplast rather than to the cytoplasm. Thus,the expression cassette can further be comprised of a chloroplasttransit peptide encoding DNA sequence operably linked between a promoterand the isolated and purified DNA segment (for a review of plastidtargeting peptides, see Heijne et al. (1989); Keegstra et al. (1989).This is exemplified by the use of the rbcS (RuBISCO) transit peptidethat targets proteins specifically to plastids.

A chloroplast transit peptide can be used. A chloroplast transit peptideis typically 40 to 70 amino acids in length and functionspost-translationally to direct a protein to the chloroplast. The transitpeptide is cleaved either during or just after import into thechloroplast to yield the mature protein. The complete copy of theisolated and purified DNA segment may contain a chloroplast transitpeptide sequence. In that case, it may not be necessary to combine anexogenously obtained chloroplast transit peptide sequence into theexpression cassette.

Chloroplast transit peptide encoding sequences can be obtained from avariety of plant nuclear genes, so long as the products of the genes areexpressed as preproteins comprising an amino terminal transit peptideand transported into chloroplast. Examples of plant gene products knownto include such transit peptide sequences include, but are not limitedto, the small subunit of ribulose biphosphate carboxylase, ferredoxin,chlorophyll a/b binding protein, chloroplast ribosomal proteins encodedby nuclear genes, certain heat shock proteins, amino acid biosyntheticenzymes such as acetolactate acid synthase,3-enolpyruvylphosphoshikimate synthase, dihydrodipicolinate synthase,and the like. Alternatively, the DNA fragment coding for the transitpeptide may be chemically synthesized either wholly or in part from theknown sequences of transit peptides such as those listed above.Furthermore, the transit peptide may compromise sequences derived fromtransit peptides from more than one source and may include a peptidesequence derived from the amino-terminal region of the mature proteinthat in its native state is linked to a transit peptide, e.g., see U.S.Pat. No. 5,510,471.

Regardless of the source of the DNA fragment coding for the transitpeptide, it should include a translation initiation codon and an aminoacid sequence that is recognized by and will function properly tofacilitate import of a polypeptide into chloroplasts of the host plant.Attention should also be given to the amino acid sequence at thejunction between the transit peptide and the protein encoded by theisolated and purified DNA segment where it is cleaved to yield themature enzyme. Certain conserved amino acid sequences have beenidentified and may serve as a guideline. Precise fusion of the transitpeptide coding sequence with the isolated and purified DNA segmentcoding sequence may require manipulation of one or both DNA sequences tointroduce, for example, a convenient restriction site. This may beaccomplished by methods including site-directed mutagenesis, insertionof chemically synthesized oligonucleotide linkers, and the like.

Once obtained, the chloroplast transit peptide sequence can beappropriately linked to the promoter and the isolated and purified DNAsegment in an expression cassette using standard methods. Briefly, aplasmid containing a promoter functional in plant cells and havingmultiple cloning sites downstream can be constructed as described inJefferson, cited supra. The chloroplast transit peptide sequence can beinserted downstream from the promoter using restriction enzymes. Theisolated and purified DNA segment can then be inserted immediatelydownstream from and in frame with the 3′ terminus of the chloroplasttransit peptide sequence so that the chloroplast transit peptide islinked to the amino terminus of the protein encoded by the isolated andpurified DNA segment. Once formed, the expression cassette can besubcloned into other plasmids or vectors.

Alternatively, targeting of the gene product to an intracellularcompartment within plant cells may also be achieved by direct deliveryof an isolated and purified DNA segment to the intracellularcompartment. For example, an expression cassette encoding a protein, thepresence of which is desired in the chloroplast, may be directlyintroduced into the chloroplast genome using the method described inU.S. Pat. No. 5,451,513.

It may be useful to target DNA itself within a cell. For example, it maybe useful to target an introduced isolated and purified DNA to thenucleus, as this may increase the frequency of transformation. Nucleartargeting sequences that function in plants are known, e.g., theAgrobacterium virD protein is known to target DNA sequences to thenucleus of a plant cell (Herrera-Estrella et al., 1990). Within thenucleus itself, it would be useful to target a gene in order to achievesite-specific integration. For example, it would be useful to have agene introduced through transformation replace an existing gene in thecell.

3. 3′ Sequences

When the expression cassette is to be introduced into a plant cell, theexpression cassette can also optionally include 3′ nontranslated plantregulatory DNA sequences that act as a signal to terminate transcriptionand allow for the polyadenylation of the resultant mRNA. The 3′nontranslated regulatory DNA sequence preferably includes from about 300to 1,000 nucleotide base pairs and contains plant transcriptional andtranslational termination sequences. Preferred 3′ elements are derivedfrom those from the nopaline synthase gene of Agrobacterium tumefaciens(Bevan et al., 1983), the terminator for the T7 transcript from theAgrobacterium tumefaciens, T-DNA and the 3′ end of the proteaseinhibitor I or II genes from potato or tomato, although other 3′elements known to those of skill in the art can also be employed. These3′ nontranslated regulatory sequences can be obtained as described inMethods in Enzymology (1987) or are already present in plasmidsavailable from commercial sources such as Clontech (Palo Alto, Calif.).The 3′ nontranslated regulatory sequences can be operably linked to the3′ terminus of the isolated and purified DNA segment by standardmethods.

4. Marker Genes

In order to improve the ability to identify transformants, one maydesire to employ one or more selectable marker genes or reporter genesas, or in addition to, the expressible isolated and purified DNAsegment(s). “Marker genes” or “reporter genes” are genes that impart adistinct phenotype to cells expressing the marker gene and thus allowsuch transformed cells to be distinguished from cells that do not havethe gene. Such genes may encode either a selectable or screenablemarker, depending on whether the marker confers a trait that one can“select” for by chemical means, i.e., through the use of a selectiveagent (e.g., a herbicide, antibiotic, or the like), or whether it issimply a “reporter” trait that one can identify through observation ortesting, i.e., by “screening” (e.g., the R-locus trait). Of course, manyexamples of suitable marker genes or reporter genes are known to the artand can be employed in the practice of the invention.

Included within the terms selectable or screenable marker genes are alsogenes that encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers that encode a secretable antigen that can be identifiedby antibody interaction, or even secretable enzymes that can be detectedby their catalytic activity. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., byELISA; and proteins that are inserted or trapped in the cell wall (e.g.,proteins that include a leader sequence such as that found in theexpression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a protein that becomes sequestered in the cell wall, and whichprotein includes a unique epitope is considered to be particularlyadvantageous. Such a secreted antigen marker would ideally employ anepitope sequence that would provide low background in plant tissue, apromoter-leader sequence that would impart efficient expression andtargeting across the plasma membrane, and would produce protein that isbound in the cell wall and yet accessible to antibodies. A normallysecreted wall protein modified to include a unique epitope would satisfyall such requirements.

One example of a protein suitable for modification in this manner isextensin, or hydroxyproline rich glycoprotein (HPRG). The use of theHPRG (Stiefel et al., 1990) is preferred as this molecule is wellcharacterized in terms of molecular biology, expression, and proteinstructure. However, any one of a variety of extensins and/orglycine-rich wall proteins (Keller et al., 1989) could be modified bythe addition of an antigenic site to create a screenable marker.

Elements of the present disclosure are exemplified in detail through theuse of particular marker genes. However in light of this disclosure,numerous other possible selectable and/or screenable marker genes willbe apparent to those of skill in the art in addition to the one setforth hereinbelow. Therefore, it will be understood that the followingdiscussion is exemplary rather than exhaustive. In light of thetechniques disclosed herein and the general recombinant techniques thatare known in the art, the present invention renders possible theintroduction of any gene, including marker genes, into a recipient cellto generate a transformed monocot.

a. Selectable Markers

Possible selectable markers for use in connection with the presentinvention include, but are not limited to, a neo gene (Potrykus et al.,1985) that codes for kanamycin resistance and can be selected for usingkanamycin, G418, and the like; a bar gene that codes for bialaphosresistance; a gene that encodes an altered EPSP synthase protein(Hinchee et al., 1988) thus conferring glyphosate resistance; anitrilase gene such as bxn from Klebsiella ozaenae that confersresistance to bromoxynil (Stalker et al., 1988); a mutant acetolactatesynthase gene (ALS) or acetoacid synthase gene (AAS) that confersresistance to imidazolinone, sulfonylurea or other ALS-inhibitingchemicals (European Patent Application 154,204); amethotrexate-resistant DHFR gene (Thillet et al., 1988); a dalapondehalogenase gene that confers resistance to the herbicide dalapon (U.S.Pat. No. 5,780,708); or a mutated anthranilate synthase gene thatconfers resistance to 5-methyl tryptophan (PCT Publication No. WO97/26366). Where a mutant EPSP synthase gene is employed, additionalbenefit may be realized through the incorporation of a suitablechloroplast transit peptide, CTP (U.S. Pat. No. 4,940,835). See also,Lundquist et al., U.S. Pat. No. 5,508,468.

An illustrative embodiment of a selectable marker gene capable of beingused in systems to select transformants is the genes that encode theenzyme phosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes (U.S. Pat. No. 5,550,318, which is incorporated byreference herein). The enzyme phosphinothricin acetyl transferase (PAT)inactivates the active ingredient in the herbicide bialaphos,phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami etal., 1986; Twell et al., 1989) causing rapid accumulation of ammonia andcell death. The success in using this selective system in conjunctionwith monocots was particularly surprising because of the majordifficulties that have been reported in transformation of cereals(Potrykus, 1989).

b. Screenable Markers or Reporter Genes

Screenable markers that may be employed include, but are not limited to,a β-glucuronidase or uidA gene (GUS) that encodes an enzyme for whichvarious chromogenic substrates are known; an R-locus gene, which encodesa product that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta et al., 1988); a β-lactamase gene(Sutcliffe, 1978), which encodes an enzyme for which various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylEgene (Zukowsky et al., 1983), which encodes a catechol dioxygenase thatcan convert chromogenic catechols; an α-amylase gene (Ikuta et al.,1990); a tyrosinase gene (Katz et al., 1983), which encodes an enzymecapable of oxidizing tyrosine to DOPA and dopaquinone which in turncondenses to form the easily detectable compound melanin; aβ-galactosidase gene, which encodes an enzyme for which there arechromogenic substrates; a luciferase (lux) gene (Ow et al., 1986), whichallows for bioluminescence detection; or even an aequorin gene (Prasheret al., 1985), which may be employed in calcium-sensitivebioluminescence detection, or a green fluorescent protein gene (Niedz etal., 1995).

Genes from the R gene complex are contemplated to be particularly usefulas screenable markers. The R gene complex in encodes a protein that actsto regulate the production of anthocyanin pigments in most seed andplant tissue. strains can have one, or as many as four, R alleles thatcombine to regulate pigmentation in a developmental and tissue specificmanner. A gene from the R gene complex was applied to transformation,because the expression of this gene in transformed cells does not harmthe cells. Thus, an R gene introduced into such cells will cause theexpression of a red pigment and, if stably incorporated, can be visuallyscored as a red sector. If a line is carries dominant alleles for genesencoding the enzymatic intermediates in the anthocyanin biosyntheticpathway (C2, A1, A2, Bz1 and Bz2), but carries a recessive allele at theR locus, transformation of any cell from that line with R will result inred pigment formation. Exemplary lines include Wisconsin 22 thatcontains the rg-Stadler allele and TR112, a K55 derivative that is r-g,b, P1. Alternatively any genotype of can be utilized if the C1 and Ralleles are introduced together.

It is further proposed that R gene regulatory regions may be employed inchimeric constructs in order to provide mechanisms for controlling theexpression of chimeric genes. More diversity of phenotypic expression isknown at the R locus than at any other locus (Coe et al., 1988). It iscontemplated that regulatory regions obtained from regions 5′ to thestructural R gene would be valuable in directing the expression ofgenes, e.g., insect resistance, drought resistance, herbicide toleranceor other protein coding regions. For the purposes of the presentinvention, it is believed that any of the various R gene family membersmay be successfully employed (e.g., P, S, Lc, etc.). However, the mostpreferred will generally be Sn (particularly Sn:bol3). Sn is a dominantmember of the R gene complex and is functionally similar to the R and Bloci in that Sn controls the tissue specific deposition of anthocyaninpigments in certain seedling and plant cells, therefore, its phenotypeis similar to R.

A further screenable marker contemplated for use in the presentinvention is firefly luciferase, encoded by the lux gene. The presenceof the lux gene in transformed cells may be detected using, for example,X-ray film, scintillation counting, fluorescent spectrophotometry,low-light video cameras, photon counting cameras or multiwellluminometry. It is also envisioned that this system may be developed forpopulational screening for bioluminescence, such as on tissue cultureplates, or even for whole plant screening.

5. Transgenes for Modification

The present invention provides methods and compositions for thetransformation of plant cells with genes in addition to, or other than,marker genes. Such transgenes will often be genes that direct theexpression of a particular protein or polypeptide product, but they mayalso be non expressible DNA segments, e.g., transposons such as Ds thatdo not direct their own tranposition. As used herein, an “expressiblegene” is any gene that is capable of being transcribed into RNA (e.g.,mRNA, antisense RNA, etc.) or translated into a protein, expressed as atrait of interest, or the like, etc., and is not limited to selectable,screenable or non-selectable marker genes. The invention alsocontemplates that, where both an expressible gene that is notnecessarily a marker gene is employed in combination with a marker gene,one may employ the separate genes on either the same or different DNAsegments for transformation. In the latter case, the different vectorsare delivered concurrently to recipient cells to maximizecotransformation.

The choice of the particular DNA segments to be delivered to therecipient cells will often depend on the purpose of the transformation.One of the major purposes of transformation of crop plants is to addsome commercially desirable, agronomically important traits to theplant. Such traits include, but are not limited to, herbicide resistanceor tolerance; insect resistance or tolerance; disease resistance ortolerance (viral, bacterial, fungal, nematode); stress tolerance and/orresistance, as exemplified by resistance or tolerance to drought, heat,chilling, freezing, excessive moisture, salt stress; oxidative stress;increased yields; food or feed content and makeup; physical appearance;male sterility; drydown; standability; prolificacy; starch properties;oil quantity and quality; and the like. One may desire to incorporateone or more genes conferring any such desirable trait or traits, suchas, for example, a gene or genes encoding herbicide resistance.

In certain embodiments, the present invention contemplates thetransformation of a recipient cell with more than one advantageoustransgene. Two or more transgenes can be supplied in a singletransformation event using either distinct transgene-encoding vectors,or using a single vector incorporating two or more gene codingsequences. For example, plasmids bearing the bar and aroA expressionunits in either convergent, divergent, colinear orientation, areconsidered to be particularly useful. Further preferred combinations arethose of an insect resistance gene, such as a Bt gene, along with aprotease inhibitor gene such as pinII, or the use of bar or anotherselectable marker gene in combination with either of the above genes. Ofcourse, any two or more transgenes of any description, such as thoseconferring herbicide, insect, disease (viral, bacterial, fungal,nematode) or drought resistance, male sterility, drydown, standability,prolificacy, starch properties, oil quantity and quality, or thoseincreasing yield or nutritional quality may be employed as desired.

a. Herbicide Resistance

The DNA segments encoding phosphinothricin acetyltransferase (bar andpat), EPSP synthase encoding genes conferring resistance to glyphosate,the glyphosate degradative enzyme gene gox encoding glyphosateoxidoreductase, deh (encoding a dehalogenase enzyme that inactivatesdalapon), herbicide resistant (e.g., sulfonylurea and imidazolinone)acetolactate synthase, and bxn genes (encoding a nitrilase enzyme thatdegrades bromoxynil) are examples of herbicide resistant genes for usein transformation. The bar and pat genes code for an enzyme,phosphinothricin acetyltransferase (PAT), which inactivates theherbicide phosphinothricin and prevents this compound from inhibitingglutamine synthetase enzymes. The enzyme 5-enolpyruvylshikimate3-phosphate synthase (EPSP Synthase), is normally inhibited by theherbicide N-(phosphonomethyl)glycine (glyphosate) in plants and mostmicroorganisms. However, genes are known that encodeglyphosate-resistant EPSP synthase enzymes, including mutated EPSPSgenes, e.g., the Salmonella typhimurium aroA CT7 mutant (Comai et al.,1985) and the naturally occurring glyphosate resistant EPSPS fromAgrobacterium, CP4 (U.S. Pat. No. 5,627,061). These genes areparticularly contemplated for use in plant transformation. The deh geneencodes the enzyme dalapon dehalogenase and confers resistance to theherbicide dalapon (U.S. Pat. No. 5,780,708). The bxn gene codes for aspecific nitrilase enzyme that converts bromoxynil to a non-herbicidaldegradation product.

b. Insect Resistance

Potential insect resistance genes that can be introduced includeBacillus thuringiensis crystal toxin genes or Bt genes (Watrud et al.,1985). Bt genes may provide resistance to economically importantlepidopteran or coleopteran pests such as European Corn Borer (ECB) andWestern Corn Rootworm, respectively. It is contemplated that preferredBt genes for use in the transformation protocols disclosed herein willbe those in which the coding sequence has been modified to effectincreased expression in plants, and more particularly, in maize. Meansfor preparing synthetic genes are well known in the art and aredisclosed in, for example, U.S. Pat. No. 5,500,365 and U.S. Pat. No.5,689,052, each of the disclosures of which are specificallyincorporated herein by reference in their entirety. Examples of suchmodified Bt toxin genes include a synthetic Bt CryIA(b) gene (Perlak etal., 1991), and the synthetic CryIA© gene termed 1800b (U.S. Pat. No.5,590,390). Some examples of other Bt toxin genes known to those ofskill in the art are given in Table 1 below.

TABLE 1 Bacillus thuringiensis δ-Endotoxin Genes^(a) New NomenclatureOld Nomenclature GenBank Accession Cry1Aa CryIA(a) M11250 Cry1AbCryIA(b) M13898 Cry1Ac CryIA(c) M11068 Cry1Ad CryIA(d) M73250 Cry1AeCryIA(e) M65252 Cry1Ba CryIB X06711 Cry1Bb ET5 L32020 Cry1Bc PEG5 Z46442Cry1Bd CryE1 U70726 Cry1Ca CryIC X07518 Cry1Cb CryIC(b) M97880 Cry1DaCryID X54160 Cry1Db PrtB Z22511 Cry1Ea CryIE X53985 Cry1Eb CryIE(b)M73253 Cry1Fa CryIF M63897 Cry1Fb PrtD Z22512 Cry1Ga PrtA Z22510 Cry1GbCryH2 U70725 Cry1Ha PrtC Z22513 Cry1Hb U35780 Cry1Ia CryV X62821 Cry1IbCryV U07642 Cry1Ja ET4 L32019 Cry1Jb ET1 U31527 Cry1K U28801 Cry2AaCryIIA M31738 Cry2Ab CryIIB M23724 Cry2Ac CryIIC X57252 Cry3A CryIIIAM22472 Cry3Ba CryIIIB X17123 Cry3Bb CryIIIB2 M89794 Cry3C CryIIID X59797Cry4A CryIVA Y00423 Cry4B CryIVB X07423 Cry5Aa CryVA(a) L07025 Cry5AbCryVA(b) L07026 Cry6A CryVIA L07022 Cry6B CryVIB L07024 Cry7Aa CryIIICM64478 Cry7Ab CryIIICb U04367 Cry8A CryIIIE U04364 Cry8B CryIIIG U04365Cry8C CryIIIF U04366 Cry9A CryIG X58120 Cry9B CryIX X75019 Cry9C CryIHZ37527 Cry10A CryIVC M12662 Cry11A CryIVD M31737 Cry11B Jeg80 X86902Cry12A CryVB L07027 Cry13A CryVC L07023 Cry14A CryVD U13955 Cry15A 34kDaM76442 Cry16A cbm71 X94146 Cry17A cbm71 X99478 Cry18A CryBP1 X99049Cry19A Jeg65 Y08920 Cyt1Aa CytA X03182 Cyt1Ab CytM X98793 Cyt2A CytBZ14147 Cyt2B CytB U52043

Protease inhibitors also may provide insect resistance (Johnson et al.,1989), and thus will have utility in plant transformation. The use of aprotease inhibitor II gene, pinII, from tomato or potato is envisionedto be particularly useful. Even more advantageous is the use of a pinIIgene in combination with a Bt toxin gene, the combined effect of whichhas been discovered to produce synergistic insecticidal activity. Othergenes that encode inhibitors of the insect's digestive system, or thosethat encode enzymes or co-factors that facilitate the production ofinhibitors, also may be useful. This group may be exemplified byoryzacystatin and amylase inhibitors such as those from wheat andbarley.

Also, genes encoding lectins may confer additional or alternativeinsecticide properties. Lectins (originally termed phytohemagglutinins)are multivalent carbohydrate-binding proteins that have the ability toagglutinate red blood cells from a range of species. Lectins have beenidentified recently as insecticidal agents with activity againstweevils, ECB and rootworm (Murdock et al., 1990; Czapla & Lang, 1990).Lectin genes contemplated to be useful include, for example, barley andwheat germ agglutinin (WGA) and rice lectins (Gatehouse et al., 1984),with WGA being preferred.

Genes controlling the production of large or small polypeptides activeagainst insects when introduced into the insect pests, such as, e.g.,lytic peptides, peptide hormones and toxins and venoms, form anotheraspect of the invention. For example, it is contemplated that theexpression of juvenile hormone esterase, directed towards specificinsect pests, also may result in insecticidal activity, or perhaps causecessation of metamorphosis (Hammock et al., 1990).

Transgenic plants expressing genes that encode enzymes that affect theintegrity of the insect cuticle form yet another aspect of theinvention. Such genes include those encoding, e.g., chitinase,proteases, lipases and also genes for the production of nikkomycin, acompound that inhibits chitin synthesis, the introduction of any ofwhich is contemplated to produce insect resistant plants. Genes thatcode for activities that affect insect molting, such as those affectingthe production of ecdysteroid UDP-glucosyl transferase, also fall withinthe scope of the useful transgenes of the present invention.

Genes that code for enzymes that facilitate the production of compoundsthat reduce the nutritional quality of the host plant to insect pestsalso are encompassed by the present invention. It may be possible, forinstance, to confer insecticidal activity on a plant by altering itssterol composition. Sterols are obtained by insects from their diet andare used for hormone synthesis and membrane stability. Therefore,alterations in plant sterol composition by expression of novel genes,e.g., those that directly promote the production of undesirable sterolsor those that convert desirable sterols into undesirable forms, couldhave a negative effect on insect growth and/or development and henceendow the plant with insecticidal activity. Lipoxygenases are naturallyoccurring plant enzymes that have been shown to exhibit anti-nutritionaleffects on insects and to reduce the nutritional quality of their diet.Therefore, further embodiments of the invention concern transgenicplants with enhanced lipoxygenase activity that may be resistant toinsect feeding.

Tripsacum dactyloides is a species of grass that is resistant to certaininsects, including corn root worm. It is anticipated that genes encodingproteins that are toxic to insects or are involved in the biosynthesisof compounds toxic to insects will be isolated from Tripsacum and thatthese novel genes will be useful in conferring resistance to insects. Itis known that the basis of insect resistance in Tripsacum is genetic,because said resistance has been transferred to Zea mays via sexualcrosses (Branson and Guss, 1972). It further is anticipated that othercereal, monocot or dicot plant species may have genes encoding proteinsthat are toxic to insects that would be useful for producing insectresistant plants.

Further genes encoding proteins characterized as having potentialinsecticidal activity also may be used as transgenes in accordanceherewith. Such genes include, for example, the cowpea trypsin inhibitor(CpTI; Hilder et al., 1987), which may be used as a rootworm deterrent;genes encoding avermectin (Avermectin and Abamectin., Campbell, 1989;Ikeda et al., 1987), which may prove particularly useful as a cornrootworm deterrent; ribosome inactivating protein genes; and even genesthat regulate plant structures. Transgenic including anti-insectantibody genes and genes that code for enzymes that can convert anon-toxic insecticide (pro-insecticide) applied to the outside of theplant into an insecticide inside the plant also are contemplated.

C. Environment or Stress Resistance

Improvement of a plants ability to tolerate various environmentalstresses such as, but not limited to, drought, excess moisture,chilling, freezing, high temperature, salt, and oxidative stress, alsocan be effected through expression of novel genes. As the ZMGRP promoterof the instant invention can be induced by such environmental stresses,genes conferring resistance to these conditions may find particular usewith this promoter.

It is proposed that benefits may be realized in terms of increasedresistance to freezing temperatures through the introduction of an“antifreeze” protein such as that of the Winter Flounder (Cutler et al.,1989) or synthetic gene derivatives thereof. Improved chilling tolerancealso may be conferred through increased expression ofglycerol-3-phosphate acetyltransferase in chloroplasts (Wolter et al.,1992). Resistance to oxidative stress (often exacerbated by conditionssuch as chilling temperatures in combination with high lightintensities) can be conferred by expression of superoxide dismutase(Gupta et al., 1993), and may be improved by glutathione reductase(Bowler et al., 1992). Such strategies may allow for tolerance tofreezing in newly emerged fields as well as extending later maturityhigher yielding varieties to earlier relative maturity zones.

It is proposed that expression of a gene encoding hemoglobin may enhancea plant's ability to assimilate and utilize oxygen, resulting in quickergermination, faster growing or maturing crops, or higher crop yields(Holmberg et al. 1997).

It is contemplated that the expression of novel genes that favorablyeffect plant water content, total water potential, osmotic potential,and turgor will enhance the ability of the plant to tolerate drought. Asused herein, the terms “drought resistance” and “drought tolerance” areused to refer to a plants increased resistance or tolerance to stressinduced by a reduction in water availability, as compared to normalcircumstances, and the ability of the plant to function and survive inlower-water environments. In this aspect of the invention it isproposed, for example, that the expression of genes encoding for thebiosynthesis of osmotically-active solutes, such as polyol compounds,may impart protection against drought. Within this class are genesencoding for mannitol-L-phosphate dehydrogenase (Lee and Saier, 1983),trehalose-6-phosphate synthase (Kaasen et al., 1992), and myo-inositolO-methyl transferase (U.S. Pat. No. 5,563,324). Through the subsequentaction of native phosphatases in the cell or by the introduction andcoexpression of a specific phosphatase, these introduced genes willresult in the accumulation of either mannitol or trehalose,respectively, both of which have been well documented as protectivecompounds able to mitigate the effects of stress. Mannitol accumulationin transgenic tobacco has been verified and preliminary results indicatethat plants expressing high levels of this metabolite are able totolerate an applied osmotic stress (Tarczynski et al., 1992, 1993).Altered water utilization in transgenic corn producing mannitol also hasbeen demonstrated (U.S. Pat. No. 5,780,709).

Similarly, the efficacy of other metabolites in protecting either enzymefunction (e.g., alanopine or propionic acid) or membrane integrity(e.g., alanopine) has been documented (Loomis et al., 1989), andtherefore expression of genes encoding for the biosynthesis of thesecompounds might confer drought resistance in a manner similar to orcomplimentary to mannitol. Other examples of naturally occurringmetabolites that are osmotically active and/or provide some directprotective effect during drought and/or desiccation include fructose,erythritol (Coxson et al., 1992), sorbitol, dulcitol (Karsten et al.,1992), glucosylglycerol (Reed et al., 1984; Erdmann et al., 1992),sucrose, stachyose (Koster and Leopold, 1988; Blackman et al., 1992),raffinose (Bernal-Lugo and Leopold, 1992), proline (Rensburg et al.,1993), glycine betaine, ononitol and pinitol (Vernon and Bohnert, 1992).Continued canopy growth and increased reproductive fitness during timesof stress will be augmented by introduction and expression of genes suchas those controlling the osmotically active compounds discussed aboveand other such compounds. Currently preferred genes that promote thesynthesis of an osmotically active polyol compound are genes that encodethe enzymes mannitol-1-phosphate dehydrogenase, trehalose-6-phosphatesynthase and myoinositol 0-methyltransferase.

It is contemplated that the expression of specific proteins also mayincrease drought tolerance. Three classes of Late Embryogenic Proteinshave been assigned based on structural similarities (see Dure et al.,1989). All three classes of LEAs have been demonstrated in maturing(i.e., desiccating) seeds. Within these 3 types of LEA proteins, theType-II (dehydrin-type) have generally been implicated in drought and/ordesiccation tolerance in vegetative plant parts (i.e., Mundy and Chua,1988; Piatkowski et al., 1990; Yamaguchi-Shinozaki et al., 1992).Recently, expression of a Type-III LEA (HVA-1) in tobacco was found toinfluence plant height, maturity and drought tolerance (Fitzpatrick,1993). In rice, expression of the HVA-1 gene influenced tolerance towater deficit and salinity (Xu et al., 1996). Expression of structuralgenes from all three LEA groups may therefore confer drought tolerance.Other types of proteins induced during water stress include thiolproteases, aldolases and transmembrane transporters (Guerrero et al.,1990), which may confer various protective and/or repair-type functionsduring drought stress. It also is contemplated that genes that effectlipid biosynthesis and hence membrane composition might also be usefulin conferring drought resistance on the plant.

Many of these genes for improving drought resistance have complementarymodes of action. Thus, it is envisaged that combinations of these genesmight have additive and/or synergistic effects in improving droughtresistance in crop plants such as, for example, corn, soybean, cotton,or wheat. Many of these genes also improve freezing tolerance (orresistance); the physical stresses incurred during freezing and droughtare similar in nature and may be mitigated in similar fashion. Benefitmay be conferred via constitutive expression of these genes, but thepreferred means of expressing these novel genes may be through the useof a turgor-induced promoter (such as the promoters for theturgor-induced genes described in Guerrero et al., 1990 and Shagan etal., 1993, which are incorporated herein by reference) or anABA-inducible promoter such as the promoter of the present invention.Inducible, spatial and temporal expression patterns of these genes mayenable plants to better withstand stress.

It is proposed that expression of genes that are involved with specificmorphological traits that allow for increased water extractions fromdrying soil would be of benefit. For example, introduction andexpression of genes that alter root characteristics may enhance wateruptake. It also is contemplated that expression of genes that enhancereproductive fitness during times of stress would be of significantvalue. For example, expression of genes that improve the synchrony ofpollen shed and receptiveness of the female flower parts, i.e., silks,would be of benefit. In addition it is proposed that expression of genesthat minimize kernel abortion during times of stress would increase theamount of grain to be harvested and hence be of value.

Given the overall role of water in determining yield, it is contemplatedthat enabling corn and other crop plants to utilize water moreefficiently, through the introduction and expression of novel genes,will improve overall performance even when soil water availability isnot limiting. By introducing genes that improve the ability of plants tomaximize water usage across a full range of stresses relating to wateravailability, yield stability or consistency of yield performance may berealized.

d. Disease Resistance

It is proposed that increased resistance to diseases may be realizedthrough introduction of genes into plants, for example, intomonocotyledonous plants such as maize. It is possible to produceresistance to diseases caused by viruses, bacteria, fungi and nematodes.It also is contemplated that control of mycotoxin producing organismsmay be realized through expression of introduced genes.

Resistance to viruses may be produced through expression of novel genes.For example, it has been demonstrated that expression of a viral coatprotein in a transgenic plant can impart resistance to infection of theplant by that virus and perhaps other closely related viruses (Cuozzo etal., 1988, Hemenway et al., 1988, Abel et al., 1986). It is contemplatedthat expression of antisense genes targeted at essential viral functionsalso may impart resistance to viruses. For example, an antisense genetargeted at the gene responsible for replication of viral nucleic acidmay inhibit replication and lead to resistance to the virus. It isbelieved that interference with other viral functions through the use ofantisense genes also may increase resistance to viruses. Similarly,ribozymes could be used in this context. Further, it is proposed that itmay be possible to achieve resistance to viruses through otherapproaches, including, but not limited to the use of satellite viruses.Examples of viral and viral-like diseases, for which one could introduceresistance to in a transgenic plant in accordance with the instantinvention, are listed below, in Table 2.

TABLE 2 Plant Virus and Virus-like Diseases DISEASE CAUSATIVE AGENTAmerican wheat striate (wheat American wheat striate mosaic virusstriate mosaic) mosaic (AWSMV) Barley stripe mosaic Barley stripe mosaicvirus (BSMV) Barley yellow dwarf Barley yellow dwarf virus (BYDV) Bromemosaic Brome mosaic virus (BMV) Cereal chlorotic Cereal chlorotic mottlevirus mottle* (CCMV) Corn chlorotic vein banding Corn chlorotic veinbanding (Brazilian mosaic)¹ virus (CCVBV) Corn lethal necrosis Viruscomplex (chlorotic mottle virus (MCMV) and dwarf mosaic vi- rus (MDMV) Aor B or Wheat streak mosaic virus (WSMV)) Cucumber mosaic Cucumbermosaic virus (CMV) Cynodon chlorotic streak*^(,1) Cynodon chloroticstreak virus (CCSV) Johnsongrass mosaic Johnsongrass mosaic virus (JGMV)bushy stunt Mycoplasma-like organism (MLO) associated chlorotic dwarfchlorotic dwarf virus (MCDV) chlorotic mottle chlorotic mottle virus(MCMV) dwarf mosaic dwarf mosaic virus (MDMV) strains A, D, E and F leaffleck leaf fleck virus (MLFV) line* line virus (MLV) mosaic (corn leafstripe, enanismo mosaic virus (MMV) rayado) mottle and chlorotic stunt¹mottle and chlorotic stunt virus* pellucid ringspot* pellucid ringspotvirus (MPRV) raya gruesa*^(,1) raya gruesa virus (MRGV) rayado fino*(fine striping disease) rayado fino virus (MRFV) red leaf and redstripe* Mollicute? red stripe* red stripe virus (MRSV) ring mottle* ringmottle virus (MRMV) rio IV* rio cuarto virus (MRCV) rough dwarf*(nanismo ruvido) rough dwarf virus (MRDV) (= Cereal tillering diseasevirus*) sterile stunt* sterile stunt virus (strains of barley yellowstriate virus) streak* streak virus (MSV) stripe (chlorotic stripe, hojablanca) stripe virus stunting*^(,1) stunting virus tassel abortion*tassel abortion virus (MTAV) vein enation* vein enation virus (MVEV)wallaby ear* wallaby ear virus (MWEV) white leaf* white leaf virus whiteline mosaic white line mosaic virus (MWLMV) Millet red leaf* Millet redleaf virus (MRLV) Northern cereal mosaic* Northern cereal mosaic virus(NCMV) Oat pseudorosette* (zakuklivanie) Oat pseudorosette virus Oatsterile dwarf* Oat sterile dwarf virus (OSDV) Rice black-streaked dwarf*Rice black-streaked dwarf virus (RBSDV) Rice stripe* Rice stripe virus(RSV) Sorghum mosaic Sorghum mosaic virus (SrMV), formerly sugarcanemosaic virus (SCMV) strains H, I and M Sugarcane Fiji disease* SugarcaneFiji disease virus (FDV) Sugarcane mosaic Sugarcane mosaic virus (SCMV)strains A, B, D, E, SC, BC, Sabi and MB (formerly MDMV-B) Veinenation*^(,1) Virus? Wheat spot mosaic¹ Wheat spot mosaic virus (WSMV)*Not known to occur naturally on corn in the United States. ¹Minor viraldisease.

It is proposed that increased resistance to diseases caused by bacteriaand fungi also may be realized through introduction of novel genes. Itis contemplated that genes encoding so-called “peptide antibiotics,”pathogenesis related (PR) proteins, toxin resistance, and proteinsaffecting host-pathogen interactions such as morphologicalcharacteristics will be useful. Peptide antibiotics are polypeptidesequences that are inhibitory to growth of bacteria and othermicroorganisms. For example, the classes of peptides referred to ascecropins and magainins inhibit growth of many species of bacteria andfungi. It is proposed that expression of PR proteins in monocotyledonousplants such as may be useful in conferring resistance to bacterialdisease. These genes are induced following pathogen attack on a hostplant and have been divided into at least five classes of proteins (Bolet al., 1990). Included amongst the PR proteins are β-1,3-glucanases,chitinases, and osmotin and other proteins that are believed to functionin plant resistance to disease organisms. Other genes have beenidentified that have antifungal properties, e.g., UDA (stinging nettlelectin), hevein (Broakaert et al., 1989; Barkai-Golan et al., 1978), andsor1 conferring resistance to photosensitizing toxins (Ehrenshaft etal., 1999). It is known that certain plant diseases are caused by theproduction of phytotoxins. It is proposed that resistance to thesediseases would be achieved through expression of a novel gene thatencodes an enzyme capable of degrading or otherwise inactivating thephytotoxin. It also is contemplated that expression of novel genes thatalter the interactions between the host plant and pathogen may be usefulin reducing the ability of the disease organism to invade the tissues ofthe host plant, e.g., an increase in the waxiness of the leaf cuticle orother morphological characteristics. Examples of bacterial and fungaldiseases, including downy mildews, for which one could introduceresistance to in a transgenic plant in accordance with the instantinvention, are listed below, in Tables 3, 4 and 5.

TABLE 3 Plant Bacterial Diseases DISEASE CAUSATIVE AGENT Bacterial leafblight and stalk rot Pseudomonas avenae subsp. avenae Bacterial leafspot Xanthomonas campestris pv. holcicola Bacterial stalk rotEnterobacter dissolvens = Erwinia dissolvens Bacterial stalk and top rotErwinia carotovora subsp. carotovora, Erwinia chrysanthemi pv. zeaeBacterial stripe Pseudomonas andropogonis Chocolate spot Pseudomonassyringae pv. coronafaciens Goss's bacterial wilt and blight Clavibactermichiganensis subsp. (leaf freckles and wilt) nebraskensis =Corynebacterium michiganense pv. nebraskense Holcus spot Pseudomonassyringae pv. syringae Purple leaf sheath Hemiparasitic bacteria + (Seeunder Fungi) Seed rot-seedling blight Bacillus subtilis Stewart'sdisease (bacterial wilt) Pantoea stewartii = Erwinia stewartii Cornstunt (achapparramiento, Spiroplasma kunkelii stunt, Mesa Central or RioGrande stunt)

TABLE 4 Plant Fungal Diseases DISEASE PATHOGEN Anthracnose leaf blightand Colletotrichum graminicola (teleomorph: anthracnose stalk rotGlomerella graminicola Politis), Glomerella tucumanensis (anamorph:Glomerella falcatum Went) Aspergillus ear and kernel rot Aspergillusflavus Link:Fr. Banded leaf and sheath spot* Rhizoctonia solani K{umlautover (uhn)} = Rhizoctonia microsclerotia J. Matz (teleomorph:Thanatephorus cucumeris) Black bundle disease Acremonium strictum W.Gams = Cephalosporium acremonium Auct. non Corda Black kernel rot*Lasiodiplodia theobromae = Botryodiplodia theobromae Borde blanco*Marasmiellus sp. Brown spot (black spot, Physoderma maydis stalk rot)Cephalosporium kernel rot Acremonium strictum = Cephalosporiumacremonium Charcoal rot Macrophomina phaseolina Corticium ear rot*Thanatephorus cucumeris = Corticium sasakii Curvularia leaf spotCurvularia clavata, C. eragrostidis, = C. maculans (teleomorph:Cochliobolus eragrostidis), Curvularia inaequalis, C. intermedia(teleomorph: Cochliobolus intermedius), Curvularia lunata (teleomorph:Cochliobolus lunatus), Curvularia pallescens (teleomorph: Cochlioboluspallescens), Curvularia senegalensis, C. tuberculata (teleomorph:Cochliobolus tuberculatus) Didymella leaf spot* Didymella exitalisDiplodia ear rot and stalk rot Diplodia frumenti (teleomorph:Botryosphaeria festucae) Diplodia ear rot, stalk rot, Diplodia maydis =Stenocarpella maydis seed rot and seedling blight Diplodia leaf spot orleaf Stenocarpella macrospora = Diplodia streak macrospora *Not known tooccur naturally on corn in the United States.

TABLE 5 Plant Downy Mildews DISEASE CAUSATIVE AGENT Brown stripe downymildew* Sclerophthora rayssiae var. zeae Crazy top downy mildewSclerophthora macrospora = Sclerospora macrospora Green ear downy mildewSclerospora graminicola (graminicola downy mildew) Java downy mildew*Peronosclerospora maydis = Sclerospora maydis Philippine downy mildew*Peronosclerospora philippinensis = Sclerospora philippinensis Sorghumdowny mildew Peronosclerospora sorghi = Sclerospora sorghi Spontaneumdowny mildew* Peronosclerospora spontanea = Sclerospora spontaneaSugarcane downy mildew* Peronosclerospora sacchari = Sclerosporasacchari Dry ear rot (cob, kernel Nigrospora oryzae (teleomorph: Khuskiaand stalk rot) oryzae) Ear rots, minor Alternaria alternata = A. tenuis,Aspergillus glaucus, A. niger, Aspergillus spp., Botrytis cinerea(teleomorph: Botryotinia fuckeliana), Cunninghamella sp., Curvulariapallescens, Doratomyces stemonitis = Cephalotrichum stemonitis, Fusariumculmorum, Gonatobotrys simplex, Pithomyces maydicus, Rhizopusmicrosporus Tiegh., R. stolonifer = R. nigricans, Scopulariopsisbrumptii. Ergot* (horse's tooth, diente de Claviceps gigantea (anamorph:Sphacelia caballo) sp.) Eyespot Aureobasidium zeae = Kabatiella zeaeFusarium ear and stalk rot Fusarium subglutinans = F. moniliforme var.subglutinans Fusarium kernel, root and stalk rot, Fusarium moniliforme(teleomorph: seed rot and seedling blight Gibberella fujikuroi) Fusariumstalk rot, seedling root rot Fusarium avenaceum (teleomorph: Gibberellaavenacea) Gibberella ear and stalk rot Gibberella zeae (anamorph:Fusarium graminearum) Gray ear rot Botryosphaeria zeae = Physalosporazeae (anamorph: Macrophoma zeae) Gray leaf spot (Cercospora leaf spot)Cercospora sorghi = C. sorghi var. maydis, C. zeae-maydisHelminthosporium root rot Exserohilum pedicellatum = Helminthosporiumpedicellatum (teleomorph: Setosphaeria pedicellata) Hormodendrum ear rotCladosporium cladosporioides = (Cladosporium rot) Hormodendrumcladosporioides, C. herbarum (teleomorph: Mycosphaerella tassiana)Hyalothyridium leaf spot* Hyalothyridium maydis Late wilt*Cephalosporium maydis Leaf spots, minor Alternaria alternata, Ascochytamaydis, A. tritici, A. zeicola, Bipolaris victoriae = Helminthosporiumvictoriae (teleomorph: Cochliobolus victoriae), C. sativus (anamorph:Bipolaris sorokiniana = H. sorokinianum = H. sativum), Epicoccum nigrum,Exserohilum prolatum = Drechslera prolata (teleomorph: Setosphaeriaprolata) Graphium penicillioides, Leptosphaeria maydis, Leptothyriumzeae, Ophiosphaerella herpotricha, (anamorph: Scolecosporiella sp.),Paraphaeosphaeria michotii, Phoma sp., Septoria zeae, S. zeicola, S.zeina Northern corn leaf blight (white Setosphaeria turcica (anamorph:blast, crown stalk rot, stripe) Exserohilum turcicum = Helminthosporiumturcicum) Northern corn leaf spot, Cochliobolus carbonum (anamorph:Helminthosporium ear rot (race 1) Bipolaris zeicola = Helminthosporiumcarbonum) Penicillium ear rot (blue eye, blue Penicillium spp., P.chrysogenum, P. mold) expansum, P. oxalicum Phaeocytostroma stalk rotand root Phaeocytostroma ambiguum, = rot Phaeocytosporella zeaePhaeosphaeria leaf spot* Phaeosphaeria maydis = Sphaerulina maydisPhysalospora ear rot (Botryosphaeria Botryosphaeria festucae =Physalospora ear rot) zeicola (anamorph: Diplodia frumenti) Purple leafsheath Hemiparasitic bacteria and fungi Pyrenochaeta stalk rot and rootrot Phoma terrestris = Pyrenochaeta terrestris Pythium root rot Pythiumspp., P. arrhenomanes, P. graminicola Pythium stalk rot Pythiumaphanidermatum = P. butleri L. Red kernel disease (ear mold, leafEpicoccum nigrum and seed rot) Rhizoctonia ear rot (sclerotial rot)Rhizoctonia zeae (teleomorph: Waitea circinata) Rhizoctonia root rot andstalk rot Rhizoctonia solani, Rhizoctonia zeae Root rots, minorAlternaria alternata, Cercospora sorghi, Dictochaeta fertilis, Fusariumacuminatum (teleomorph: Gibberella acuminata), F. equiseti (teleomorph:G. intricans), F. oxysporum, F. pallidoroseum, F. poae, F. roseum, G.cyanogena, (anamorph: F. sulphureum), Microdochium bolleyi, Mucor sp.,Periconia circinata, Phytophthora cactorum, P. drechsleri, P. nicotianaevar. parasitica, Rhizopus arrhizus Rostratum leaf spot Setosphaeriarostrata, (anamorph: (Helminthosporium leaf disease, ear Exserohilumrostratum = and stalk rot) Helminthosporium rostratum) Rust, common cornPuccinia sorghi Rust, southern corn Puccinia polysora Rust, tropicalcorn Physopella pallescens, P. zeae = Angiopsora zeae Sclerotium earrot* (southern blight) Sclerotium rolftii Sacc. (teleomorph: Atheliarolfsii) Seed rot-seedling blight Bipolaris sorokiniana, B. zeicola =Helminthosporium carbonum, Diplodia maydis, Exserohilum pedicillatum,Exserohilum turcicum = Helminthosporium turcicum, Fusarium avenaceum, F.culmorum, F. moniliforme, Gibberella zeae (anamorph: F. graminearum),Macrophomina phaseolina, Penicillium spp., Phomopsis sp., Pythium spp.,Rhizoctonia solani, R. zeae, Sclerotium rolfsii, Spicaria sp.Selenophoma leaf spot* Selenophoma sp. Sheath rot Gaeumannomycesgraminis Shuck rot Myrothecium gramineum Silage mold Monascus purpureus,M. ruber Smut, common Ustilago zeae = U. maydis) Smut, falseUstilaginoidea virens Smut, head Sphacelotheca reiliana = Sporisoriumholci-sorghi Southern corn leaf blight and stalk Cochliobolusheterostrophus (anamorph: rot Bipolaris maydis = Helminthosporiummaydis) Southern leaf spot Stenocarpella macrospora = Diplodiamacrospora Stalk rots, minor Cercospora sorghi, Fusarium episphaeria, F.merismoides, F. oxysporum Schlechtend, F. poae, F. roseum, F. solani(teleomorph: Nectria haematococca), F. tricinctum, Mariannaea elegans,Mucor sp., Rhopographus zeae, Spicaria sp. Storage rots Aspergillusspp., Penicillium spp. and other fungi Tar spot* Phyllachora maydisTrichoderma ear rot and root rot Trichoderma viride = T. lignorumteleomorph: Hypocrea sp. White ear rot, root and stalk rot Stenocarpellamaydis = Diplodia zeae Yellow leaf blight Ascochyta ischaemi,Phyllosticta maydis (teleomorph: Mycosphaerella zeae-maydis) Zonate leafspot Gloeocercospora sorghi *Not known to occur naturally on corn in theUnited States.

Plant parasitic nematodes are a cause of disease in many plants,including cereal plants such as maize, barley, wheat, rye and rice. Itis proposed that it would be possible to make plants resistant to theseorganisms through the expression of novel gene products. It isanticipated that control of nematode infestations would be accomplishedby altering the ability of the nematode to recognize or attach to a hostplant and/or enabling the plant to produce nematicidal compounds,including but not limited to proteins. It is known that certainendotoxins derived from Bacillus thuringiensis are nematicidal (Bottjeret al., 1985; U.S. Pat. No. 5,831,011). Examples of nematode-associatedplant diseases, for which one could introduce resistance to in atransgenic plant in accordance with the invention are given below, inTable 6.

TABLE 6 Parasitic Nematodes DISEASE PATHOGEN Awl Dolichodorus spp, D.heterocephalus Bulb and stem (Europe) Ditylenchus dipsaci BurrowingRadopholus similis Cyst Heterodera avenae, H. zeae, Punctoderachalcoensis Dagger Xiphinema spp., X. americanum, X. mediterraneum Falseroot-knot Nacobbus dorsalis Lance, Columbia Hoplolaimus columbus LanceHoplolaimus spp., H. galeatus Lesion Pratylenchus spp., P. brachyurus,P. crenatus, P. hexincisus, P. neglectus, P. penetrans, P. scribneri, P.thornei, P. zeae Needle Longidorus spp., L. breviannulatus RingCriconemella spp., C. ornata Root-knot Meloidogyne spp., M. chitwoodi,M. incognita, M. javanica Spiral Helicotylenchus spp. Sting Belonolaimusspp., B. longicaudatus Stubby-root Paratrichodorus spp., P. christiei,P. minor, Quinisulcius acutus, Trichodorus spp. Stunt Tylenchorhynchusdubius

e. Mycotoxin Reduction/Elimination

Production of mycotoxins, including aflatoxin and fumonisin, by fungiassociated with monocotyledonous plants such as cereal plants, includingmaize, barley, wheat, rye or rice, is a significant factor in renderingthe grain not useful. These fungal organisms do not cause diseasesymptoms and/or interfere with the growth of the plant, but they producechemicals (mycotoxins) that are toxic to animals. It is contemplatedthat inhibition of the growth of these fungi would reduce the synthesisof these toxic substances and therefore reduce grain losses due tomycotoxin contamination. It also is proposed that it may be possible tointroduce novel genes into monocotyledonous plants such as that wouldinhibit synthesis of the mycotoxin. Further, it is contemplated thatexpression of a novel gene that encodes an enzyme capable of renderingthe mycotoxin nontoxic would be useful in order to achieve reducedmycotoxin contamination of grain. The result of any of the abovemechanisms would be a reduced presence of mycotoxins on grain.

f. Grain Composition or Quality

Genes may be introduced into monocotyledonous plants, particularlycommercially important cereals, such as maize, barley, wheat, rye orrice, to improve the grain for which the cereal is primarily grown. Awide range of novel transgenic plants produced in this manner may beenvisioned depending on the particular end use of the grain.

The largest use of grain is for feed or food. Introduction of genes thatalter the composition of the grain may greatly enhance the feed or foodvalue. The primary components of grain are starch, protein, and oil.Each of these primary components of grain may be improved by alteringits level or composition. Several examples may be mentioned forillustrative purposes, but in no way provide an exhaustive list ofpossibilities.

The protein of cereal grains including maize, barley, wheat, rye andrice is suboptimal for feed and food purposes especially when fed tomonogastric animals such as pigs, poultry, and humans. The protein isdeficient in several amino acids that are essential in the diet of thesespecies, requiring the addition of supplements to the grain. Limitingessential amino acids may include lysine, methionine, tryptophan,threonine, valine, arginine, and histidine. Some amino acids becomelimiting only after corn is supplemented with other inputs for feedformulations. For example, when corn is supplemented with soybean mealto meet lysine requirements methionine becomes limiting. The levels ofthese essential amino acids in seeds and grain may be elevated bymechanisms that include, but are not limited to, the introduction ofgenes to increase the biosynthesis of the amino acids, decrease thedegradation of the amino acids, increase the storage of the amino acidsin proteins, direct the storage of amino acids in proteins comprising anutritionally enhanced balance of amino acids, or increase transport ofthe amino acids to the seeds or grain.

One mechanism for increasing the biosynthesis of the amino acids is tointroduce genes that deregulate the amino acid biosynthetic pathwayssuch that the plant can no longer adequately control the levels that areproduced. This may be done by deregulating or bypassing steps in theamino acid biosynthetic pathway that are normally regulated by levels ofthe amino acid end product of the pathway. Examples include theintroduction of genes that encode deregulated versions of the enzymesaspartokinase or dihydrodipicolinic acid (DHDP)-synthase for increasinglysine and threonine production, and anthranilate synthase forincreasing tryptophan production. Reduction of the catabolism of theamino acids may be accomplished by introduction of DNA sequences thatreduce or eliminate the expression of genes encoding enzymes thatcatalyze steps in the catabolic pathways such as the enzymelysine-ketoglutarate reductase. It is anticipated that it may bedesirable to target expression of genes relating to amino acidbiosynthesis to the endosperm or embryo of the seed. More preferably,the gene will be targeted to the embryo. It will also be preferable forgenes encoding proteins involved in amino acid biosynthesis to targetthe protein to a plastid using a plastid transit peptide sequence.

The protein composition of the grain may be altered to improve thebalance of amino acids in a variety of ways including elevatingexpression of native proteins, decreasing expression of those with poorcomposition, changing the composition of native proteins, or introducinggenes encoding entirely new proteins possessing superior composition.Examples may include the introduction of DNA that decreases theexpression of members of the zein family of storage proteins. This DNAmay encode ribozymes or antisense sequences directed to impairingexpression of zein proteins or expression of regulators of zeinexpression such as the opaque-2 gene product. It also is proposed thatthe protein composition of the grain may be modified through thephenomenon of co-suppression, i.e., inhibition of expression of anendogenous gene through the expression of an identical structural geneor gene fragment introduced through transformation (Goring et al., 1991;PCT Publication No. WO 98/26064). Additionally, the introduced DNA mayencode enzymes that degrade zeins. The decreases in zein expression thatare achieved may be accompanied by increases in proteins with moredesirable amino acid composition or increases in other major seedconstituents such as starch. Alternatively, a chimeric gene may beintroduced that comprises a coding sequence for a native protein ofadequate amino acid composition such as for one of the globulin proteinsor 10 kD delta zein or 20 kD delta zein or 27 kD gamma zein of and apromoter or other regulatory sequence designed to elevate expression ofsaid protein. The coding sequence of the gene may include additional orreplacement codons for essential amino acids. Further, a coding sequenceobtained from another species, or, a partially or completely syntheticsequence encoding a completely unique peptide sequence designed toenhance the amino acid composition of the seed may be employed. It isanticipated that it may be preferable to target expression of thesetransgenes encoding proteins with superior composition to the endospermof the seed.

The introduction of genes that alter the oil content of the grain may beof value. Increases in oil content may result in increases inmetabolizable-energy-content and density of the seeds for use in feedand food. The introduced genes may encode enzymes that remove or reducerate-limitations or regulated steps in fatty acid or lipid biosynthesis.Such genes may include, but are not limited to, those that encodeacetyl-CoA carboxylase, ACP-acyltransferase, β-ketoacyl-ACP synthase,plus other well known fatty acid biosynthetic activities. Otherpossibilities are genes that encode proteins that do not possessenzymatic activity such as acyl carrier protein. Genes may be introducedthat alter the balance of fatty acids present in the oil providing amore healthful or nutritive feedstuff. The introduced DNA also mayencode sequences that block expression of enzymes involved in fatty acidbiosynthesis, altering the proportions of fatty acids present in thegrain such as described below. Some other examples of genes specificallycontemplated by the inventors for use in creating transgenic plants withaltered oil composition traits include 2-acetyltransferase, oleosin,pyruvate dehydrogenase complex, acetyl CoA synthetase, ATP citratelyase, ADP-glucose pyrophosphorylase and genes of thecarnitine-CoA-acetyl-CoA shuttles. It is anticipated that expression ofgenes related to oil biosynthesis will be targeted to the plastid, usinga plastid transit peptide sequence and preferably expressed in the seedembryo.

Genes may be introduced that enhance the nutritive value of the starchcomponent of the grain, for example by increasing the degree ofbranching, resulting in improved utilization of the starch, for example,in cows by delaying its metabolism. It is contemplated that alterationof starch structure may improve the wet milling properties of grain ormay produce a starch composition with improved qualities for industrialutilization. It is anticipated that expression of genes related tostarch biosynthesis will preferably be targeted to the endosperm of theseed.

Besides affecting the major constituents of the grain, genes may beintroduced that affect a variety of other nutritive, processing, orother quality aspects of the grain as used for feed or food. Forexample, pigmentation of the grain may be increased or decreased.Enhancement and stability of yellow pigmentation is desirable in someanimal feeds and may be achieved by introduction of genes that result inenhanced production of xanthophylls and carotenes by eliminatingrate-limiting steps in their production. Such genes may encode alteredforms of the enzymes phytoene synthase, phytoene desaturase, or lycopenesynthase. Alternatively, unpigmented white corn is desirable forproduction of many food products and may be produced by the introductionof DNA that blocks or eliminates steps in pigment production pathways.

Most of the phosphorous content of the grain is in the form of phytate,a form of phosphate storage that is not metabolized by monogastricanimals. Therefore, in order to increase the availability of seedphosphate, it is anticipated that one will desire to decrease the amountof phytate in seed and increase the amount of free phosphorous. It isanticipated that one can decrease the expression or activity of one ofthe enzymes involved in the synthesis of phytate. For example,suppression of expression of the gene encoding inositol phosphatesynthetase (INOPS) may lead to an overall reduction in phytateaccumulation. It is anticipated that antisense or sense suppression ofgene expression may be used. Alternatively, one may express a gene inseed that will be activated, e.g., by pH, in the gastric system of amonogastric animal and will release phosphate from phytate, e.g.,phytase. It is further contemplated that one may provide an alternatestorage form for phosphate in the grain, wherein the storage form ismore readily utilized by a monogastric animal.

Feed or food comprising primarily maize or other cereal grains possessesinsufficient quantities of vitamins and must be supplemented to provideadequate nutritive value. Introduction of genes that enhance vitaminbiosynthesis in seeds may be envisioned including, for example, vitaminsA, E, B₁₂, choline, and the like. Maize grain also does not possesssufficient mineral content for optimal nutritive value. Genes thataffect the accumulation or availability of compounds containingphosphorus, sulfur, calcium, manganese, zinc, and iron among otherswould be valuable. An example may be the introduction of a gene thatreduced phytic acid production or encoded the enzyme phytase thatenhances phytic acid breakdown. These genes would increase levels ofavailable phosphate in the diet, reducing the need for supplementationwith mineral phosphate.

Numerous other examples of improvement of other plants for feed and foodpurposes might be described. The improvements may not even necessarilyinvolve the grain, but may, for example, improve the value of the plantsfor silage. Introduction of DNA to accomplish this might includesequences that alter lignin production such as those that result in the“brown midrib” phenotype associated with superior feed value for cattle.

In addition to direct improvements in feed or food value, genes also maybe introduced that improve the processing of plant material and improvethe value of the products resulting from the processing. For example,the primary method of processing maize is via wetmilling that may beimproved though the expression of novel genes that increase theefficiency and reduce the cost of processing such as by decreasingsteeping time.

Improving the value of wetmilling products may include altering thequantity or quality of starch, oil, corn gluten meal, or the componentsof corn gluten feed. Elevation of starch may be achieved through theidentification and elimination of rate limiting steps in starchbiosynthesis or by decreasing levels of the other components of thegrain resulting in proportional increases in starch. An example of theformer may be the introduction of genes encoding ADP-glucosepyrophosphorylase enzymes with altered regulatory activity or that areexpressed at higher level. Examples of the latter may include selectiveinhibitors of, for example, protein or oil biosynthesis expressed duringlater stages of kernel development.

The properties of starch may be beneficially altered by changing theratio of amylose to amylopectin, the size of the starch molecules, ortheir branching pattern. Through these changes a broad range ofproperties may be modified that include, but are not limited to, changesin gelatinization temperature, heat of gelatinization, clarity of filmsand pastes, Theological properties, and the like. To accomplish thesechanges in properties, genes that encode granule-bound or soluble starchsynthase activity or branching enzyme activity may be introduced aloneor combination. DNA such as antisense constructs also may be used todecrease levels of endogenous activity of these enzymes. The introducedgenes or constructs may possess regulatory sequences that time theirexpression to specific intervals in starch biosynthesis and starchgranule development. Furthermore, it may be worthwhile to introduce andexpress genes that result in the in vivo derivatization, or othermodification, of the glucose moieties of the starch molecule. Thecovalent attachment of any molecule may be envisioned, limited only bythe existence of enzymes that catalyze the derivatizations and theaccessibility of appropriate substrates in the starch granule. Examplesof important derivations may include the addition of functional groupssuch as amines, carboxyls, or phosphate groups that provide sites forsubsequent in vitro derivatizations or affect starch properties throughthe introduction of ionic charges. Examples of other modifications mayinclude direct changes of the glucose units such as loss of hydroxylgroups or their oxidation to aldehyde or carboxyl groups.

Oil is another product of wetmilling of corn, the value of which may beimproved by introduction and expression of genes. The quantity of oilthat can be extracted by wetmilling may be elevated by approaches asdescribed for feed and food above. Oil properties also may be altered toimprove its performance in the production and use of cooking oil,shortenings, lubricants or other oil-derived products or improvement ofits health attributes when used in the food-related applications. Novelfatty acids also may be synthesized that upon extraction can serve asstarting materials for chemical syntheses. The changes in oil propertiesmay be achieved by altering the type, level, or lipid arrangement of thefatty acids present in the oil. This in turn may be accomplished by theaddition of genes that encode enzymes that catalyze the synthesis ofnovel fatty acids and the lipids possessing them or by increasing levelsof native fatty acids while possibly reducing levels of precursors.Alternatively, DNA sequences may be introduced that slow or block stepsin fatty acid biosynthesis resulting in the increase in precursor fattyacid intermediates. Genes that might be added include desaturases,epoxidases, hydratases, dehydratases, and other enzymes that catalyzereactions involving fatty acid intermediates. Representative examples ofcatalytic steps that might be blocked include the desaturations fromstearic to oleic acid and oleic to linolenic acid resulting in therespective accumulations of stearic and oleic acids. Another example isthe blockage of elongation steps resulting in the accumulation of C₈ toC₁₂ saturated fatty acids.

Improvements in the other major corn wetmilling products, corn glutenmeal and corn gluten feed, also may be achieved by the introduction ofgenes to obtain novel corn plants. Representative possibilities includebut are not limited to those described above for improvement of food andfeed value.

In addition, it may further be considered that a plant, such as maize orother monocots, may be used for the production or manufacturing ofuseful biological compounds that were either not produced at all, or notproduced at the same level, in the plant previously. The novel plantsproducing these compounds are made possible by the introduction andexpression of genes by transformation methods. The vast array ofpossibilities include but are not limited to any biological compoundthat is presently produced by any organism such as proteins, nucleicacids, primary and intermediary metabolites, carbohydrate polymers, etc.The compounds may be produced by the plant, extracted upon harvestand/or processing, and used for any presently recognized useful purposesuch as pharmaceuticals, fragrances, and industrial enzymes to name afew.

Further possibilities to exemplify the range of grain traits orproperties potentially encoded by introduced genes in transgenic plantsinclude grain with less breakage susceptibility for export purposes orlarger grit size when processed by dry milling through introduction ofgenes that enhance γ-zein synthesis, popcorn with improved poppingquality and expansion volume through genes that increase pericarpthickness, corn with whiter grain for food uses though introduction ofgenes that effectively block expression of enzymes involved in pigmentproduction pathways, and improved quality of alcoholic beverages orsweet corn through introduction of genes that affect flavor such as theshrunken 1 gene (encoding sucrose synthase) or shrunken 2 gene (encodingADPG pyrophosphorylase) for sweet corn.

g. Plant Agronomic Characteristics

Two of the factors determining where crop plants can be grown are theaverage daily temperature during the growing season and the length oftime between frosts. Within the areas where it is possible to grow aparticular crop, there are varying limitations on the maximal time thecrop has available to grow to maturity and be harvested. For example, tobe grown in a particular area is selected for its ability to mature anddry down to harvestable moisture content within the required period oftime with maximum possible yield. Therefore, plants, including maize orother cereals, of varying maturities are developed for different growinglocations. Apart from the need to dry down sufficiently to permitharvest, it is desirable to have maximal drying take place in the fieldto minimize the amount of energy required for additional post-harvestdrying. Also, the more readily the grain can dry down, the more timethere is available for growth and seed maturation. It is considered thatgenes that influence maturity and/or dry down can be identified andintroduced into corn or other plants using transformation techniques tocreate new varieties adapted to different growing locations or the samegrowing location, but having improved yield to moisture ratio atharvest. Expression of genes that are involved in regulation of plantdevelopment may be especially useful, e.g., the liguleless and roughsheath genes that have been identified in corn.

It is contemplated that genes may be introduced into plants that wouldimprove standability and other plant growth characteristics. Expressionof novel genes that confer stronger stalks, improved root systems, orprevent or reduce ear droppage would be of great value to the farmer. Itis proposed that introduction and expression of genes that increase thetotal amount of photoassimilate available by, for example, increasinglight distribution and/or interception would be advantageous. Inaddition, the expression of genes that increase the efficiency ofphotosynthesis and/or the leaf canopy would further increase gains inproductivity. It is contemplated that expression of a phytochrome genein plants, including maize, may be advantageous. Expression of such agene may reduce apical dominance, confer semidwarfism on a plant, andincrease shade tolerance (U.S. Pat. No. 5,268,526). Such approacheswould allow for increased plant populations in the field.

Delay of late season vegetative senescence would increase the flow ofassimilate into the grain and thus increase yield. It is proposed thatoverexpression of genes within a plant such as maize that are associatedwith “stay green” or the expression of any gene that delays senescencewould be advantageous. For example, a nonyellowing mutant has beenidentified in Festuca pratensis (Davies et al., 1990). Expression ofthis gene as well as others may prevent premature breakdown ofchlorophyll and thus maintain canopy function.

h. Nutrient Utilization

The ability to utilize available nutrients may be a limiting factor ingrowth of monocotyledonous plants such as maize, barley, wheat, rye orrice. It is proposed that it would be possible to alter nutrient uptake,tolerate pH extremes, mobilization through the plant, storage pools, andavailability for metabolic activities by the introduction of novelgenes. These modifications would allow a plant such as maize, barley,wheat, rye or rice to more efficiently utilize available nutrients. Itis contemplated that an increase in the activity of, for example, anenzyme that is normally present in the plant and involved in nutrientutilization would increase the availability of a nutrient. An example ofsuch an enzyme would be phytase. It further is contemplated thatenhanced nitrogen utilization by a plant is desirable. Expression of aglutamate dehydrogenase gene in plants such as maize, e.g., E. coli gdhAgenes, may lead to increased fixation of nitrogen in organic compounds.Furthermore, expression of gdhA in a plant may lead to enhancedresistance to the herbicide glufosinate by incorporation of excessammonia into glutamate, thereby detoxifying the ammonia. It also iscontemplated that expression of a novel gene may make a nutrient sourceavailable that was previously not accessible, e.g., an enzyme thatreleases a component of nutrient value from a more complex molecule,perhaps a macromolecule.

i. Male Sterility

Male sterility is useful in the production of hybrid seed. It isproposed that male sterility may be produced through expression of novelgenes. For example, it has been shown that expression of genes thatencode proteins that interfere with development of the maleinflorescence and/or gametophyte result in male sterility. Chimericribonuclease genes that express in the anthers of transgenic tobacco andoilseed rape have been demonstrated to lead to male sterility (Marianiet al., 1990).

A number of mutations were discovered in maize that confer cytoplasmicmale sterility. One mutation in particular, referred to as T cytoplasm,also correlates 10 with sensitivity to Southern corn leaf blight. A DNAsequence, designated TURF-13 (Levings, 1990), was identified thatcorrelates with T cytoplasm. It is proposed that it would be possiblethrough the introduction of TURF-13 via transformation, to separate malesterility from disease sensitivity. As it is necessary to be able torestore male fertility for breeding purposes and for grain production,it is proposed that genes encoding restoration of male fertility alsomay be introduced.

j. Negative Selectable Markers

Introduction of genes encoding traits that can be selected against maybe useful for eliminating undesirable linked genes. It is contemplatedthat when two or more genes are introduced together by cotransformationthat the genes will be linked together on the host chromosome. Forexample, a gene encoding Bt that confers insect resistance on the plantmay be introduced into a plant together with a bar gene that is usefulas a selectable marker and confers resistance to the herbicide LIBERTY®on the plant. However, it may not be desirable to have an insectresistant plant that also is resistant to the herbicide LIBERTY®. It isproposed that one also could introduce an antisense bar coding regionthat is expressed in those tissues where one does not want expression ofthe bar gene product, e.g., in whole plant parts. Hence, although thebar gene is expressed and is useful as a selectable marker, it is notuseful to confer herbicide resistance on the whole plant. The barantisense gene is a negative selectable marker.

It also is contemplated that negative selection is necessary in order toscreen a population of transformants for rare homologous recombinantsgenerated through gene targeting. For example, a homologous recombinantmay be identified through the inactivation of a gene that was previouslyexpressed in that cell. The antisense construct for neomycinphosphotransferase II (NPT II) has been investigated as a negativeselectable marker in tobacco (Nicotiana tabacum) and Arabidopsisthaliana (Xiang. and Guerra, 1993). In this example, both sense andantisense NPT II genes are introduced into a plant throughtransformation and the resultant plants are sensitive to the antibiotickanamycin. An introduced gene that integrates into the host cellchromosome at the site of the antisense NPT II gene, and inactivates theantisense gene, will make the plant resistant to kanamycin and otheraminoglycoside antibiotics. Therefore, rare, site-specific recombinantsmay be identified by screening for antibiotic resistance. Similarly, anygene, native to the plant or introduced through transformation, thatwhen inactivated confers resistance to a compound, may be useful as anegative selectable marker.

It is contemplated that negative selectable markers also may be usefulin other ways. One application is to construct transgenic lines in whichone could select for transposition to unlinked sites. In the process oftagging it is most common for the transposable element to move to agenetically linked site on the same chromosome. A selectable marker forrecovery of rare plants in which transposition has occurred to anunlinked locus would be useful. For example, the enzyme cytosinedeaminase may be useful for this purpose. In the presence of this enzymethe non-phytotoxic compound 5-fluorocytosine is converted to5-fluorouracil, which is toxic to plant and animal cells. If atransposable element is linked to the gene for the enzyme cytosinedeaminase, one may select for transposition to unlinked sites byselecting for transposition events in which the resultant plant is nowresistant to 5-fluorocytosine. The parental plants and plants containingtranspositions to linked sites will remain sensitive to5-fluorocytosine. Resistance to 5-fluorocytosine is due to loss of thecytosine deaminase gene through genetic segregation of the transposableelement and the cytosine deaminase gene. Other genes that encodeproteins that render the plant sensitive to a certain compound will alsobe useful in this context. For example, T-DNA gene 2 from Agrobacteriumtumefaciens encodes a protein that catalyzes the conversion ofα-naphthalene acetamide (NAM) to α-naphthalene acetic acid (NAA) rendersplant cells sensitive to high concentrations of NAM (Depicker et al.,1988).

It also is contemplated that negative selectable markers may be usefulin the construction of transposon tagging lines. For example, by markingan autonomous transposable element such as Ac, Master Mu, or En/Spn witha negative selectable marker, one could select for transformants inwhich the autonomous element is not stably integrated into the genome.It is proposed that this would be desirable, for example, when transientexpression of the autonomous element is desired to activate in trans thetransposition of a defective transposable element, such as Ds, butstable integration of the autonomous element is not desired. Thepresence of the autonomous element may not be desired in order tostabilize the defective element, i.e., prevent it from furthertransposing. However, it is proposed that if stable integration of anautonomous transposable element is desired in a plant the presence of anegative selectable marker may make it possible to eliminate theautonomous element during the breeding process.

k. Non-Protein-Expressing Sequences

DNA may be introduced into plants for the purpose of expressing RNAtranscripts that function to affect plant phenotype yet are nottranslated into protein. Two examples are antisense RNA and RNA withribozyme activity. Both may serve possible functions in reducing oreliminating expression of native or introduced plant genes. However, asdetailed below, DNA need not be expressed to effect the phenotype of aplant.

1. Antisense RNA

Genes may be constructed or isolated, which when transcribed, produceantisense RNA that is complementary to all or part(s) of a targetedmessenger RNA(s). The antisense RNA reduces production of thepolypeptide product of the messenger RNA. The polypeptide product may beany protein encoded by the plant genome. The aforementioned genes willbe referred to as antisense genes. An antisense gene may thus beintroduced into a plant by transformation methods to produce a noveltransgenic plant with reduced expression of a selected protein ofinterest. For example, the protein may be an enzyme that catalyzes areaction in the plant. Reduction of the enzyme activity may reduce oreliminate products of the reaction that include any enzymaticallysynthesized compound in the plant such as fatty acids, amino acids,carbohydrates, nucleic acids and the like. Alternatively, the proteinmay be a storage protein, such as a zein, or a structural protein, thedecreased expression of which may lead to changes in seed amino acidcomposition or plant morphological changes respectively. Thepossibilities cited above are provided only by way of example and do notrepresent the full range of applications.

2. Ribozymes

Genes also may be constructed or isolated, which when transcribed,produce RNA enzymes (ribozymes) that can act as endoribonucleases andcatalyze the cleavage of RNA molecules with selected sequences. Thecleavage of selected messenger RNAs can result in the reduced productionof their encoded polypeptide products. These genes may be used toprepare novel transgenic plants that possess them. The transgenic plantsmay possess reduced levels of polypeptides including, but not limitedto, the polypeptides cited above.

Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987;Forster and Symons, 1987). For example, a large number of ribozymesaccelerate phosphoester transfer reactions with a high degree ofspecificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes.

Several different ribozyme motifs have been described with RNA cleavageactivity (Symons, 1992). Examples include sequences from the Group Iself splicing introns including Tobacco Ringspot Virus (Prody et al.,1986), Avocado Sunblotch Viroid (Palukaitis et al., 1979; Symons, 1981),and Lucerne Transient Streak Virus (Forster and Symons, 1987). Sequencesfrom these and related viruses are referred to as hammerhead ribozymebased on a predicted folded secondary structure.

Other suitable ribozymes include sequences from RNase P with RNAcleavage activity (Yuan et al., 1992, Yuan and Altman, 1994, U.S. Pat.Nos. 5,168,053 and 5,624,824), hairpin ribozyme structures(Berzal-Herranz et al., 1992; Chowrira et al., 1993) and Hepatitis Deltavirus based ribozymes (U.S. Pat. No. 5,625,047). The general design andoptimization of ribozyme directed RNA cleavage activity has beendiscussed in detail (Haseloff and Gerlach, 1988, Symons, 1992, Chowriraet al., 1994; Thompson et al., 1995).

The other variable on ribozyme design is the selection of a cleavagesite on a given target RNA. Ribozymes are targeted to a given sequenceby virtue of annealing to a site by complimentary base pairinteractions. Two stretches of homology are required for this targeting.These stretches of homologous sequences flank the catalytic ribozymestructure defined above. Each stretch of homologous sequence can vary inlength from 7 to 15 nucleotides. The only requirement for defining thehomologous sequences is that, on the target RNA, they are separated by aspecific sequence that is the cleavage site. For hammerhead ribozyme,the cleavage site is a dinucleotide sequence on the target RNA is auracil (U) followed by either an adenine, cytosine or uracil (A, C or U)(Perriman et al., 1992; Thompson et al., 1995). The frequency of thisdinucleotide occurring in any given RNA is statistically 3 out of 16.Therefore, for a given target messenger RNA of 1000 bases, 187dinucleotide cleavage sites are statistically possible.

Designing and testing ribozymes for efficient cleavage of a target RNAis a process well known to those skilled in the art. Examples ofscientific methods for designing and testing ribozymes are described byChowrira et al., (1994) and Lieber and Strauss (1995), each incorporatedby reference. The identification of operative and preferred sequencesfor use in down regulating a given gene is simply a matter of preparingand testing a given sequence, and is a routinely practiced “screening”method known to those of skill in the art.

3. Induction of Gene Silencing

It also is possible that genes may be introduced to produce noveltransgenic plants that have reduced expression of a native gene productby the mechanism of co-suppression. It has been demonstrated in tobacco,tomato, petunia, and corn (Goring et al., 1991; Smith et al., 1990;Napoli et al., 1990; van der Krol et al., 1990; PCT Publication No. WO98/26064) that expression of the sense transcript of a native gene willreduce or eliminate expression of the native gene in a manner similar tothat observed for antisense genes. The introduced gene may encode all orpart of the targeted native protein but its translation may not berequired for reduction of levels of that native protein.

4. Non-RNA-Expressing Sequences

DNA elements including those of transposable elements such as Ds, Ac, orMu, may be inserted into a gene to cause mutations. These DNA elementsmay be inserted in order to inactivate (or activate) a gene and thereby“tag” a particular trait. In this instance the transposable element doesnot cause instability of the tagged mutation, because the utility of theelement does not depend on its ability to move in the genome. Once adesired trait is tagged, the introduced DNA sequence may be used toclone the corresponding gene, e.g., using the introduced DNA sequence asa PCR primer target sequence together with PCR gene cloning techniques(Shapiro, 1983; Dellaporta et al., 1988). Once identified, the entiregene(s) for the particular trait, including control or regulatoryregions where desired, may be isolated, cloned and manipulated asdesired. The utility of DNA elements introduced into an organism forpurposes of gene tagging is independent of the DNA sequence and does notdepend on any biological activity of the DNA sequence, i.e.,transcription into RNA or translation into protein. The sole function ofthe DNA element is to disrupt the DNA sequence of a gene.

It is contemplated that unexpressed DNA sequences, including novelsynthetic sequences, could be introduced into cells as proprietary“labels” of those cells and plants and seeds thereof. It would not benecessary for a label DNA element to disrupt the function of a geneendogenous to the host organism, as the sole function of this DNA wouldbe to identify the origin of the organism. For example, one couldintroduce a unique DNA sequence into a plant and this DNA element wouldidentify all cells, plants, and progeny of these cells as having arisenfrom that labeled source. It is proposed that inclusion of label DNAswould enable one to distinguish proprietary germplasm or germplasmderived from such, from unlabelled germplasm.

Another possible element that may be introduced is a matrix attachmentregion element (MAR), such as the chicken lysozyme A element (Stief,1989), which can be positioned around an expressible gene of interest toeffect an increase in overall expression of the gene and diminishposition dependent effects upon incorporation into the plant genome(Stief et al., 1989; Phi-Van et al., 1990).

5. Other Sequences

An expression cassette of the invention can also be further compriseplasmid DNA. Plasmid vectors include additional DNA sequences thatprovide for easy selection, amplification, and transformation of theexpression cassette in prokaryotic and eukaryotic cells, e.g.,pUC-derived vectors such as pUC8, pUC9, pUC18, pUC19, pUC23, pUC119, andpUC120, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors,or pBS-derived vectors. The additional DNA sequences include origins ofreplication to provide for autonomous replication of the vector,selectable marker genes, preferably encoding antibiotic or herbicideresistance, unique multiple cloning sites providing for multiple sitesto insert DNA sequences or genes encoded in the expression cassette, andsequences that enhance transformation of prokaryotic and eukaryoticcells.

Another vector that is useful for expression in both plant andprokaryotic cells is the binary Ti plasmid (as disclosed in U.S. Pat.No. 4,940,838) as exemplified by vector pGA582. This binary Ti plasmidvector has been previously characterized by An (1989) and is availablefrom Dr. An. This binary Ti vector can be replicated in prokaryoticbacteria such as E. coli and Agrobacterium. The Agrobacterium plasmidvectors can be used to transfer the expression cassette to plant cells.The binary Ti vectors preferably include the nopaline T DNA right andleft borders to provide for efficient plant cell transformation, aselectable marker gene, unique multiple cloning sites in the T borderregions, the co/E1 replication of origin and a wide host range replicon.The binary Ti vectors carrying an expression cassette of the inventioncan be used to transform both prokaryotic and eukaryotic cells, but ispreferably used to transform plant cells.

In certain embodiments, it is contemplated that one may wish to employreplication-competent viral vectors in monocot transformation. Suchvectors include, for example, wheat dwarf virus (WDV) “shuttle” vectors,such as pW1-11 and PW1-GUS (Ugaki et al., 1991). These vectors arecapable of autonomous replication in cells as well as E. coli, and assuch may provide increased sensitivity for detecting DNA delivered totransgenic cells. A replicating vector may also be useful for deliveryof genes flanked by DNA sequences from transposable elements such as Ac,Ds, or Mu. It has been proposed (Laufs el al., 1990) that transpositionof these elements within the genome requires DNA replication. It is alsocontemplated that transposable elements would be useful for introducingDNA fragments lacking elements necessary for selection and maintenanceof the plasmid vector in bacteria, e.g., antibiotic resistance genes andorigins of DNA replication. It is also proposed that use of atransposable element such as Ac, Ds, or Mu would actively promoteintegration of the desired DNA and hence increase the frequency ofstably transformed cells.

Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNAsegments for use in transforming such cells will, of course, generallycomprise the isolated and purified cDNA(s), isolated and purified DNA(s)or genes that one desires to introduce into the cells. These DNAconstructs can further include structures such as promoters, enhancers,polylinkers, or even regulatory genes as desired. The DNA segment orgene chosen for cellular introduction will often encode a protein thatwill be expressed in the resultant recombinant cells, such as willresult in a screenable or selectable trait and/or that will impart animproved phenotype to the regenerated plant. However, this may notalways be the case, and the present invention also encompassestransgenic plants incorporating non-expressed transgenes.

III. Methods for Plant Transformation

Suitable methods for plant transformation for use with the currentinvention are believed to include virtually any method by which DNA canbe introduced into a cell, such as by direct delivery of DNA such as byPEG-mediated transformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), byelectroporation (U.S. Pat. No. 5,384,253, specifically incorporatedherein by reference in its entirety), by agitation with silicon carbidefibers (Kaeppler et al., 1990; U.S. Pat. No. 5,302,523, specificallyincorporated herein by reference in its entirety; and U.S. Pat. No.5,464,765, specifically incorporated herein by reference in itsentirety), by Agrobacterium-mediated transformation (U.S. Pat. No.5,591,616 and U.S. Pat. No. 5,563,055; both specifically incorporatedherein by reference) and by acceleration of DNA coated particles (U.S.Pat. No. 5,550,318; U.S. Pat. No. 5,538,877; and U.S. Pat. No.5,538,880; each specifically incorporated herein by reference in itsentirety), etc. Through the application of techniques such as these,cells as well as those of virtually any other plant species may bestably transformed, and these cells developed into transgenic plants. Incertain embodiments, acceleration methods are preferred and include, forexample, microprojectile bombardment and the like.

A. Electroporation

Where one wishes to introduce DNA by means of electroporation, it iscontemplated that the method of Krzyzek et al. (U.S. Pat. No. 5,384,253,incorporated herein by reference in its entirety) will be particularlyadvantageous. In this method, certain cell wall-degrading enzymes, suchas pectin-degrading enzymes, are employed to render the target recipientcells more susceptible to transformation by electroporation thanuntreated cells. Alternatively, recipient cells are made moresusceptible to transformation by mechanical wounding.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wounding in acontrolled manner. Examples of some species that have been transformedby electroporation of intact cells include (U.S. Pat. No. 5,384,253;Rhodes et al., 1995; D'Halluin et al., 1992), wheat (Zhou et al., 1993),tomato (Hou and Lin, 1996), soybean (Christou et al., 1987) and tobacco(Lee et al., 1989).

One also may employ protoplasts for electroporation transformation ofplants (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon-derivedprotoplasts is described by Dhir and Widholm in PCT Publication No. WO92/17598 (specifically incorporated herein by reference). Other examplesof species for which protoplast transformation has been describedinclude barley (Lazerri, 1995), sorghum (Battraw et al., 1991),(Bhattacharjee et al., 1997), wheat (He et al., 1994) and tomato(Tsukada, 1989).

B. Microprojectile Bombardment

A preferred method for delivering transforming DNA segments to plantcells in accordance with the invention is microprojectile bombardment(U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No.5,610,042; and U.S. Pat. No. 5,590,390; each of which is specificallyincorporated herein by reference in its entirety). In this method,particles may be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, platinum, and preferably, gold. It is contemplated that insome instances DNA precipitation onto metal particles would not benecessary for DNA delivery to a recipient cell using microprojectilebombardment. However, it is contemplated that particles may contain DNArather than be coated with DNA. Hence, it is proposed that DNA-coatedparticles may increase the level of DNA delivery via particlebombardment but are not, in and of themselves, necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System(BioRad, Hercules, Calif.), which can be used to propel particles coatedwith DNA or cells through a screen, such as a stainless steel or Nytexscreen, onto a filter surface covered with monocot plant cells culturedin suspension. The screen disperses the particles so that they are notdelivered to the recipient cells in large aggregates. It is believedthat a screen intervening between the projectile apparatus and the cellsto be bombarded reduces the size of projectiles aggregate and maycontribute to a higher frequency of transformation by reducing thedamage inflicted on the recipient cells by projectiles that are toolarge.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. Examples of species forwhich have been transformed by microprojectile bombardment includemonocot species such as (U.S. Pat. No. 5,590,390), barley (Ritala etal., 1994; Hensgens et al., 1993), wheat (U.S. Pat. No. 5,563,055,specifically incorporated herein by reference in its entirety), rice(Hensgens et al., 1993), oat (Torbet et al., 1995; Torbet et al., 1998),rye (Hensgens et al., 1993), sugarcane (Bower et al., 1992), and sorghum(Casa et al., 1993; Hagio et al., 1991); as well as a number of dicotsincluding tobacco (Tomes et al., 1990; Buising and Benbow, 1994),soybean (U.S. Pat. No. 5,322,783, specifically incorporated herein byreference in its entirety), sunflower (Knittel et al. 1994), peanut(Singsit et al., 1997), cotton (McCabe and Martinell, 1993), tomato (VanEck et al. 1995), and legumes in general (U.S. Pat. No. 5,563,055,specifically incorporated herein by reference in its entirety).

C. Agrobacterium-mediated Transformation

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described by Fraley etal., (1985), Rogers et al., (1987) and U.S. Pat. No. 5,563,055,specifically incorporated herein by reference in its entirety.

Agrobacterium-mediated transformation is most efficient indicotyledonous plants and is the preferable method for transformation ofdicots, including Arabidopsis, tobacco, tomato, and potato. Indeed,while Agrobacterium-mediated transformation has been routinely used withdicotyledonous plants for a number of years, it has only recently becomeapplicable to monocotyledonous plants. Advances inAgrobacterium-mediated transformation techniques have now made thetechnique applicable to nearly all monocotyledonous plants. For example,Agrobacterium-mediated transformation techniques have now been appliedto rice (Hiei et al., 1997; Zhang et al., 1997; U.S. Pat. No. 5,591,616,specifically incorporated herein by reference in its entirety), wheat(McCormac et al., 1998), barley (Tingay et al., 1997; McCormac et al.,1998), and rice (Ishida et al., 1996).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described(Rogers et al., 1987) have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes. Inaddition, Agrobacterium containing both armed and disarmed Ti genes canbe used for the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

D. Other Transformation Methods

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Omirulleh et al., 1993; Frommet al., 1986; Uchimiya et al., 1986; Callis et al., 1987; Marcotte etal., 1988).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastshave been described (Toriyama et al., 1986; Yamada et al., 1986;Abdullah et al., 1986; Omirulleh et al., 1993 and U.S. Pat. No.5,508,184; each specifically incorporated herein by reference in itsentirety). Examples of the use of direct uptake transformation of cerealprotoplasts include transformation of rice (Ghosh-Biswas et al., 1994),sorghum (Battraw and Hall, 1991), barley (Lazerri, 1995), oat (Zheng andEdwards, 1990) and maize (Omirulleh et al., 1993).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, 1989). Also,silicon carbide fiber-mediated transformation may be used with orwithout protoplasting (Kaeppler, 1990; Kaeppler et al., 1992; U.S. Pat.No. 5,563,055, specifically incorporated herein by reference in itsentirety). Transformation with this technique is accomplished byagitating silicon carbide fibers together with cells in a DNA solution.DNA passively enters as the cell are punctured. This technique has beenused successfully with, for example, the monocot cereals (U.S. Pat. No.5,590,390, specifically incorporated herein by reference in itsentirety; Thompson, 1995) and rice (Nagatani, 1997).

IV. Optimization of Microprojectile Bombardment

For microprojectile bombardment transformation in accordance with thecurrent invention, both physical and biological parameters may beoptimized. Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the flight andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells before andimmediately after bombardment, such as the osmotic adjustment of targetcells to help alleviate the trauma associated with bombardment, theorientation of an immature embryo or other target tissue relative to theparticle trajectory, and also the nature of the transforming DNA, suchas linearized DNA or intact supercoiled plasmids. It is believed thatpre-bombardment manipulations are especially important for successfultransformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust various ofthe bombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas DNA concentration, gap distance, flight distance, tissue distance,and helium pressure. It further is contemplated that the grade of heliummay effect transformation efficiency. For example, differences intransformation efficiencies may be witnessed between bombardments usingindustrial grade (99.99% pure) or ultra pure helium (99.999% pure),although it is not currently clear that is more advantageous for use inbombardment. One also may optimize the trauma reduction factors (TRFs)by modifying conditions that influence the physiological state of therecipient cells and that may therefore influence transformation andintegration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation.

A. Physical Parameters

1. Gap Distance

The variable nest (macro holder) can be adjusted to vary the distancebetween the rupture disk and the macroprojectile, i.e., the gapdistance. This distance can be varied from 0 to 2 cm. The predictedeffects of a shorter gap are an increase of velocity of both the macro-and microprojectiles, an increased shock wave (which leads to tissuesplattering and increased tissue trauma), and deeper penetration ofmicroprojectiles. Longer gap distances would have the opposite effectsbut may increase viability and therefore the total number of recoveredstable transformants.

2. Flight Distance

The fixed nest (contained within the variable nest) can be variedbetween 0.5 and 2.25 cm in predetermined 0.5 cm increments by theplacement of spacer rings to adjust the flight path traversed by themacroprojectile. Short flight paths allow for greater stability of themacroprojectile in flight but reduce the overall velocity of themicroprojectiles. Increased stability in flight increases, for example,the number of centered GUS foci. Greater flight distances (up to apoint) increase velocity but also increase instability in flight. Basedon observations, it is recommended that bombardments typically be donewith a flight path length of about 1.0 cm to 1.5 cm.

3. Tissue Distance

Placement of tissue within the gun chamber can have significant effectson microprojectile penetration. Increasing the flight path of themicroprojectiles will decrease velocity and trauma associated with theshock wave. A decrease in velocity also will result in shallowerpenetration of the microprojectiles.

4. Helium Pressure

By manipulation of the type and number of rupture disks, pressure can bevaried between 400 and 2000 psi within the gas acceleration tube.Optimum pressure for stable transformation has been determined to bebetween 1000 and 1200 psi.

5. Coating of Microprojectiles.

For microprojectile bombardment, one will attach (i.e., “coat”) DNA tothe microprojectiles such that it is delivered to recipient cells in aform suitable for transformation thereof. In this respect, at least someof the transforming DNA must be available to the target cell fortransformation to occur, while at the same time during delivery the DNAmust be attached to the microprojectile. Therefore, availability of thetransforming DNA from the microprojectile may comprise the physicalreversal of bonds between transforming DNA and the microprojectilefollowing delivery of the microprojectile to the target cell. This neednot be the case, however, as availability to a target cell may occur asa result of breakage of unbound segments of DNA or of other moleculesthat comprise the physical attachment to the microprojectile.Availability may further occur as a result of breakage of bonds betweenthe transforming DNA and other molecules, which are either directly orindirectly attached to the microprojectile. It further is contemplatedthat transformation of a target cell may occur by way of directrecombination between the transforming DNA and the genomic DNA of therecipient cell. Therefore, as used herein, a “coated” microprojectilewill be one that is capable of being used to transform a target cell, inthat the transforming DNA will be delivered to the target cell, yet willbe accessible to the target cell such that transformation may occur.

Any technique for coating microprojectiles that allows for delivery oftransforming DNA to the target cells may be used. Methods for coatingmicroprojectiles that have been demonstrated to work well with thecurrent invention have been specifically disclosed herein. DNA may bebound to microprojectile particles using alternative techniques,however. For example, particles may be coated with streptavidin and DNAend labeled with long chain thiol cleavable biotinylated nucleotidechains. The DNA adheres to the particles due to the streptavidin-biotininteraction, but is released in the cell by reduction of the thiollinkage through reducing agents present in the cell.

Alternatively, particles may be prepared by functionalizing the surfaceof a gold oxide particle, providing free amine groups. DNA, having astrong negative charge, binds to the functionalized particles.Furthermore, charged particles may be deposited in controlled arrays onthe surface of mylar flyer disks used in the PDS-1000 Biolistics device,thereby facilitating controlled distribution of particles delivered totarget tissue.

As disclosed above, it further is proposed, that the concentration ofDNA used to coat microprojectiles may influence the recovery oftransformants containing a single copy of the transgene. For example, alower concentration of DNA may not necessarily change the efficiency ofthe transformation, but may instead increase the proportion of singlecopy insertion events. In this regard, approximately 1 ng to 2000 ng oftransforming DNA may be used per each 1.8 mg of startingmicroprojectiles. In other embodiments of the invention, approximately2.5 ng to 1000 ng, 2.5 ng to 750 ng, 2.5 ng to 500 ng, 2.5 ng to 250 ng,2.5 ng to 100 ng, or 2.5 ng to 50 ng of transforming DNA may be used pereach 1.8 mg of starting microprojectiles.

Various other methods also may be used to increase transformationefficiency and/or increase the relative proportion of low-copytransformation events. For example, the inventors contemplateend-modifying transforming DNA with alkaline phosphatase or an agentthat will blunt DNA ends prior to transformation. Still further, aninert carrier DNA may be included with the transforming DNA, therebylowering the effective transforming DNA concentration without loweringthe overall amount of DNA used. These techniques are further describedin U.S. patent application Ser. No. 08/995,451, filed Dec. 22, 1997,U.S. Pat. No. 6,153,811, the disclosure now of which is specificallyincorporated herein by reference in its entirety.

B. Biological Parameters

Culturing conditions and other factors can influence the physiologicalstate of the target cells and may have profound effects ontransformation and integration efficiencies. First, the act ofbombardment could stimulate the production of ethylene, which could leadto senescence of the tissue. The addition of antiethylene compoundscould increase transformation efficiencies. Second, it is proposed thatcertain points in the cell cycle may be more appropriate for integrationof introduced DNA. Hence synchronization of cell cultures may enhancethe frequency of production of transformants. For example,synchronization may be achieved using cold treatment, amino acidstarvation, or other cell cycle-arresting agents. Third, the degree oftissue hydration also may contribute to the amount of trauma associatedwith bombardment as well as the ability of the microprojectiles topenetrate cell walls.

The position and orientation of an embryo or other target tissuerelative to the particle trajectory also may be important. For example,the PDS-1000 biolistics device does not produce a uniform spread ofparticles over the surface of a target petri dish. The velocity ofparticles in the center of the plate is higher than the particlevelocity at further distances from the center of the petri dish.Therefore, it is advantageous to situate target tissue on the petri dishsuch as to avoid the center of the dish, referred to by some as the“zone of death.” Furthermore, orientation of the target tissue withregard to the trajectory of targets also can be important. It iscontemplated that it is desirable to orient the tissue most likely toregenerate a plant toward the particle stream. For example, thescutellum of an immature embryo comprises the cells of greatestembryogenic potential and therefore should be oriented toward theparticle stream.

It also has been reported that slightly plasmolyzed yeast cells allowincreased transformation efficiencies (Armaleo et al., 1990). It washypothesized that the altered osmotic state of the cells helped toreduce trauma associated with the penetration of the microprojectile.Additionally, the growth and cell cycle stage may be important withrespect to transformation.

1. Osmotic Adjustment

It has been suggested that osmotic pre-treatment could potentiallyreduce bombardment associated injury as a result of the decreased turgorpressure of the plasmolyzed cell. In a previous study, the number ofcells transiently expressing GUS increased following subculture intoboth fresh medium and osmotically adjusted medium (U.S. Pat. No.5,590,390, specifically incorporated herein by reference in itsentirety). Pretreatment times of 90 minutes showed higher numbers of GUSexpressing foci than shorter times. Cells incubated in 500 mOSM/kgmedium for 90 minutes showed an approximately 3.5 fold increase intransient GUS foci than the control. Preferably, immature embryos areprecultured for 4-5 hours prior to bombardment on culture mediumcontaining 12% sucrose. A second culture on 12% sucrose is performed for16-24 hours following bombardment. Alternatively, type II cells arepretreated on 0.2M mannitol for 3-4 hours prior to bombardment. It iscontemplated that pretreatment of cells with other osmotically activesolutes for a period of 1-6 hours also may be desirable.

2. Plasmid Configuration

In some instances, it will be desirable to deliver DNA to cells thatdoes not contain DNA sequences necessary for maintenance of the plasmidvector in the bacterial host, e.g., E. coli, such as antibioticresistance genes, including but not limited to ampicillin, kanamycin,and tetracycline resistance, and prokaryotic origins of DNA replication.In such case, a DNA fragment containing the transforming DNA may bepurified prior to transformation. An exemplary method of purification isgel electrophoresis on a 1.2% low melting temperature agarose gel,followed by recovery from the agarose gel by melting gel slices in a6-10 fold excess of Tris-EDTA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA,70° C.-72° C.); frozen and thawed (37° C.); and the agarose pelleted bycentrifugation. A Qiagen Q-100 column then may be used for purificationof DNA. For efficient recovery of DNA, the flow rate of the column maybe adjusted to 40 ml/hr.

Isolated DNA fragments can be recovered from agarose gels using avariety of electroelution techniques, enzyme digestion of the agarose,or binding of DNA to glass beads (e.g., Gene Clean). In addition, HPLCand/or use of magnetic particles may be used to isolate DNA fragments.As an alternative to isolation of DNA fragments, a plasmid vector can bedigested with a restriction enzyme and this DNA delivered to cellswithout prior purification of the expression cassette fragment.

V. Recipient Cells for Transformation

Tissue culture requires media and controlled environments. “Media”refers to the numerous nutrient mixtures that are used to grow cells invitro, that is, outside of the intact living organism. The mediumusually is a suspension of various categories of ingredients (salts,amino acids, growth regulators, sugars, buffers) that are required forgrowth of most cell types. However, each specific cell type requires aspecific range of ingredient proportions for growth, and an even morespecific range of formulas for optimum growth. Rate of cell growth alsowill vary among cultures initiated with the array of media that permitgrowth of that cell type.

Nutrient media is prepared as a liquid, but this may be solidified byadding the liquid to materials capable of providing a solid support.Agar is most commonly used for this purpose. Bactoagar, Hazelton agar,Gelrite, and Gelgro are specific types of solid support that aresuitable for growth of plant cells in tissue culture.

Some cell types will grow and divide either in liquid suspension or onsolid media. As disclosed herein, plant cells will grow in suspension oron solid medium, but regeneration of plants from suspension culturestypically requires transfer from liquid to solid media at some point indevelopment. The type and extent of differentiation of cells in culturewill be affected not only by the type of media used and by theenvironment, for example, pH, but also by whether media is solid orliquid. Table 7 illustrates the composition of various media useful forcreation of recipient cells and for plant regeneration.

Recipient cell targets include, but are not limited to, meristem cells,Type I, Type II, and Type III callus, immature embryos and gametic cellssuch as microspores, pollen, sperm and egg cells. It is contemplatedthat any cell from which a fertile plant may be regenerated is useful asa recipient cell. Type I, Type II, and Type III callus may be initiatedfrom tissue sources including, but not limited to, immature embryos,seedling apical meristems, microspores and the like. Those cells thatare capable of proliferating as callus also are recipient cells forgenetic transformation. The present invention provides techniques fortransforming immature embryos and subsequent regeneration of fertiletransgenic plants. Transformation of immature embryos obviates the needfor long term development of recipient cell cultures. Pollen, as well asits precursor cells, microspores, may be capable of functioning asrecipient cells for genetic transformation, or as vectors to carryforeign DNA for incorporation during fertilization. Direct pollentransformation would obviate the need for cell culture. Meristematiccells (i.e., plant cells capable of continual cell division andcharacterized by an undifferentiated cytological appearance, normallyfound at growing points or tissues in plants such as root tips, stemapices, lateral buds, etc.) may represent another type of recipientplant cell. Because of their undifferentiated growth and capacity fororgan differentiation and totipotency, a single transformed meristematiccell could be recovered as a whole transformed plant. In fact, it isproposed that embryogenic suspension cultures may be an in vitromeristematic cell system, retaining an ability for continued celldivision in an undifferentiated state, controlled by the mediaenvironment.

Cultured plant cells that can serve as recipient cells for transformingwith desired DNA segments may be any plant cells including maize cells,and more specifically, cells from Zea mays L. Somatic cells are ofvarious types. Embryogenic cells are one example of somatic cells thatmay be induced to regenerate a plant through embryo formation.Non-embryogenic cells are those that typically will not respond in sucha fashion. An example of non-embryogenic cells are certain Black MexicanSweet (BMS) corn cells.

The development of embryogenic calli and suspension cultures useful inthe context of the present invention, e.g., as recipient cells fortransformation, has been described in U.S. Pat. No. 5,134,074; and U.S.Pat. No. 5,489,520; each of which is incorporated herein by reference inits entirety.

Certain techniques may be used that enrich recipient cells within a cellpopulation. For example, Type II callus development, followed by manualselection and culture of friable, embryogenic tissue, generally resultsin an enrichment of recipient cells for use in, microprojectiletransformation. Suspension culturing, particularly using the mediadisclosed herein, may improve the ratio of recipient to non-recipientcells in any given population. Manual selection techniques that can beemployed to select recipient cells may include, e.g., assessing cellmorphology and differentiation, or may use various physical orbiological means. Cryopreservation also is a possible method ofselecting for recipient cells.

Manual selection of recipient cells, e.g., by selecting embryogeniccells from the surface of a Type II callus, is one means that may beused in an attempt to enrich for recipient cells prior to culturing(whether cultured on solid media or in suspension). The preferred cellsmay be those located at the surface of a cell cluster, and may furtherbe identifiable by their lack of differentiation, their size and densecytoplasm. The preferred cells will generally be those cells that areless differentiated, or not yet committed to differentiation. Thus, onemay wish to identify and select those cells that are cytoplasmicallydense, relatively unvacuolated with a high nucleus to cytoplasm ratio(e.g., determined by cytological observations), small in size (e.g.,10-20 μm), and capable of sustained divisions and somatic proembryoformation.

It is proposed that other means for identifying such cells also may beemployed. For example, through the use of dyes, such as Evan's blue,which are excluded by cells with relatively non-permeable membranes,such as embryogenic cells, and taken up by relatively differentiatedcells such as root-like cells and snake cells (so-called due to theirsnake-like appearance).

Other possible means of identifying recipient cells include the use ofisozyme markers of embryogenic cells, such as glutamate dehydrogenase,which can be detected by cytochemical stains (Fransz et al., 1989).However, it is cautioned that the use of isozyme markers includingglutamate dehydrogenase may lead to some degree of false positives fromnon-embryogenic cells such as rooty cells that nonetheless have arelatively high metabolic activity.

A. Culturing Cells to be Recipients for Transformation

The ability to prepare and cryopreserve cultures of plant cells isimportant to certain aspects of the present invention, in that itprovides a means for reproducibly and successfully preparing cells fortransformation. A variety of different types of media have beenpreviously developed and may be employed in carrying out various aspectsof the invention. The following table, Table 7, sets forth thecomposition of the media preferred by the inventor for carrying outthese aspects of the invention.

TABLE 7 Tissue Culture Media That are Used for Type II CallusDevelopment, Development of Suspension Cultures and Regeneration ofPlant Cells (Particularly Cells) MEDIA BASAL SUC- OTHER COMPONENTS** NO.MEDIUM ROSE pH (Amount/L) 7 MS* 2% 6.0 .25 mg thiamine .5 mg BAP .5 mgNAA Bactoagar 10 MS 2% 6.0 .25 mg thiamine 1 mg BAP 1 mg 2,4-D 400 mgL-proline Bactoagar 19 MS 2% 6.0 .25 mg thiamine .25 mg BAP .25 mg NAABactoagar 20 MS 3% 6.0 .25 mg thiamine 1 mg BAP 1 mg NAA Bactoagar 52 MS2% 6.0 .25 mg thiamine 1 mg 2,4-D 10⁻⁷M ABA BACTOAGAR 101 MS 3% 6.0 MSvitamins 100 mg myo-inositol Bactoagar 105 MS — 3.5 0.04 mg NAA 3 mg BAP1 mg thiamine.HCl 0.5 mg niacin 0.91 mg L-asparagine monohydrate 100 mgmyo-inositol 100 mg casein hydrolysate 1.4 g L-proline 20 g sorbitol 2.0g Gelgro 110 MS 6% 5.8 1 mg thiamine.HCl 1 mg niacin 3.6 g Gelgro 142 MS6% 6.0 MS vitamins 5 mg BAP 0.186 mg NAA 0.175 mg IAA 0.403 mg 2IPBactoagar 157 MS 6% 6.0 MS vitamins 100 mg myo-inositol Bactoagar 163 MS3% 6.0 MS vitamins 3.3 mg dicamba 100 mg myo-inositol Bactoagar 171 MS3% 6.0 MS vitamins .25 mg 2,4-D 10 mg BAP 100 mg myo-inositol Bactogar173 MS 6% 6.0 MS vitamins 5 mg BAP .186 mg NAA .175 mg IAA .403 mg 2IP10⁻⁷M ABA 200 mg myo-inositol Bactoagar 177 MS 3% 6.0 MS vitamins .25 mg2,4-D 10 mg BAP 10⁻⁷M ABA 100 mg myo-inositol Bactoagar 185 MS — 5.8 3mg BAP .04 mg NAA RT vitamins 1.65 mg thiamine 1.38 g L-proline 20 gsorbitol Bactoagar 189 MS — 5.8 3 mg BAP .04 mg NAA .5 mg niacin 800 mgL-asparagine 100 mg casamino acids 20 g sorbitol 1.4 g L-proline 100 mgmyo-inositol Gelgro 201 N6 2% 5.8 N6 vitamins 2 mg L-glycine 1 mg 2,4-D100 mg casein hydrolysate 2.9 g L-proline Gelgro 205 N6 2% 5.8 N6vitamins 2 mg L-glycine .5 mg 2,4-D 100 mg casein hydrolysate 2.9 gL-proline Gelgro 209 N6 6% 5.8 N6 vitamins 2 mg L-glycine 100 mg caseinhydrolysate 0.69 g L-proline Bactoagar 210 N6 3% 5.5 N6 vitamins 2 mg2,4-D 250 mg Ca pantothenate 100 mg myo-inositol 790 mg L-asparagine 100mg casein hydrolysate 1.4 g L-proline Hazelton agar**** 2 mg L-glycine211 N6 2% 5.8 1 mg 2,4-D 0.5 mg niacin 1.0 mg thiamine 0.91 gL-asparagine 100 mg myo-inositol 0.5 g MES 100 mg/L casein hydrolysate1.6 g MgCl₂-6 H₂O 0.69 g L-proline 2 g Gelgro 212 N6 3% 5.5 N6 vitamins2 mg L-glycine 2 mg 2,4-D 250 mg Ca pantothenate 100 mg myo-inositol 100mg casein hydrolysate 1.4 g L-proline Hazelton agar**** 227 N6 2% 5.8 N6vitamins 2 mg L-glycine 13.2 mg dicamba 100 mg casein hydrolysate 2.9 gL-proline Gelgro 273 (also, N6 2% 5.8 N6 vitamins 201V, 236S, 2 mgL-glycine 201D, 2071, 1 mg 2,4-D 2366, 16.9 mg AgNO₃ 201SV, 100 mgcasein hydrolysate 2377, and 2.9 g L-proline 201BV) 279 N6 2% 5.8 3.3 mgdicamba 1 mg thiamine .5 mg niacin 800 mg L-asparagine 100 mg caseinhydrolysate 100 mg myoinositol 1.4 g L-proline Gelgro**** 288 N6 3% 3.3mg dicamba 1 mg thiamine .5 mg niacin .8 g L-asparagine 100 mgmyo-inosital 1.4 g L-proline 100 mg casein hydrolysate 16.9 mg AgNO₃Gelgro 401 MS 3% 6.0 3.73 mg Na₂EDTA .25 mg thiamine 1 mg 2,4-D 2 mg NAA200 mg casein hydrolysate 500 mg K₂SO₄ 400 mg KH₂PO₄ 100 mg myo-inositol402 MS 3% 6.0 3.73 mg Na₂EDTA .25 mg thiamine 1 mg 2,4-D 200 mg caseinhydrolysate 2.9 g L-proline 500 mg K₂SO₄ 400 mg KH₂PO₄ 100 mgmyo-inositol 409 MS 3% 6.0 3.73 mg Na₂EDTA .25 mg thiamine 9.9 mgdicamba 200 mg casein hydrolysate 2.9 g L-proline 500 mg K₂SO₄ 400 mgKH₂PO₄ 100 mg myo-inositol 501 Clark's 2% 5.7 Me- dium*** 607 1/2 × MS3% 5.8 1 mg thiamine 1 mg niacin Gelrite 615 MS 3% 6.0 MS vitamins 6 mgBAP 100 mg myo-inositol Bactoagar 617 1/2 × MS 1.5%   6.0 MS vitamins 50mg myo-inositol Bactoagar 708 N6 2% 5.8 N6 vitamins 2 mg L-glycine 1.5mg 2,4-D 200 mg casein hydrolysate 0.69 g L-proline Gelrite 721 N6 2%5.8 3.3 mg dicamba 1 mg thiamine .5 mg niacin 800 mg L-asparagine 100 mgmyo-inositol 100 mg casein hydrolysate 1.4 g L-proline 54.65 g mannitolGelgro 726 N6 3% 5.8 3.3 mg dicamba .5 mg niacin 1 mg thiamine 800 mgL-asparagine 100 mg myo-inositol 100 mg casein hydrolysate 1.4 gL-proline 727 N6 3% 5.8 N6 vitamins 2 mg L-glycine 9.9 mg dicamba 100 mgcasein hydrolysate 2.9 g L-proline Gelgro 728 N6 3% 5.8 N6 vitamins 2 mgL-glycine 9.9 mg dicamba 16.9 mg AgNO₃ 100 mg casein hydrolysate 2.9 gL-proline Gelgro 734 N6 2% 5.8 N6 vitamins 2 mg L-glycine 1.5 mg 2,4-D14 g Fe sequestreene (replaces Fe-EDTA) 200 mg casein hydrolyste 0.69 gL-proline Gelrite 735 N6 2% 5.8 1 mg 2,4-D .5 mg niacin .91 gL-asparagine 100 mg myo-inositol 1 mg thiamine .5 g MES .75 g MgCl₂ 100mg casein hydrolysate 0.69 g L-proline Gelgro 2004  N6 3% 5.8 1 mgthiamine 0.5 mg niacin 3.3 mg dicamba 17 mg AgNO₃ 1.4 g L-proline 0.8 gL-asparagine 100 mg casein hydrolysate 100 mg myo-inositol Gelrite 2008 N6 3% 5.8 1 mg thiamine 0.5 mg niacin 3.3 mg dicamba 1.4 g L-proline 0.8g L-asparagine Gelrite *Basic MS medium described in Murashige and Skoog(1962). This medium is typically modified by decreasing the NH₄NO₃ from1.64 g/l to 1.55 g/l, and omitting the pyridoxine HCl, nicotinic acid,myo-inositol and glycine. **NAA = Napthol Acetic Acid IAA = IndoleAcetic Acid 2-IP = 2, isopentyl adenine 2,4-D = 2,4-Dichlorophenoxyacetic Acid BAP = 6-Benzyl aminopurine ABA = abscisicacid ***Basic medium described in Clark (1982) ****These media may bemade with or without solidifying agent.

A number of exemplary cultures that may be used for transformation havebeen developed and are disclosed in U.S. Pat. No. 5,590,390, thedisclosure of which is specifically incorporated herein by reference.

B. Media

In certain embodiments of the current invention, recipient cells may be.selected following growth in culture. Where employed, cultured cells maybe grown either on solid supports or in the form of liquid suspensions.In either instance, nutrients may be provided to the cells in the formof media, and environmental conditions controlled. There are many typesof tissue culture media comprised of various amino acids, salts, sugars,growth regulators and vitamins. Most of the media employed in thepractice of the invention will have some similar components (see Table7), but may differ in the composition and proportions of theiringredients depending on the particular application envisioned. Forexample, various cell types usually grow in more than one type of media,but will exhibit different growth rates and different morphologies,depending on the growth media. In some media, cells survive but do notdivide.

Various types of media suitable for culture of plant cells previouslyhave been described. Examples of these media include, but are notlimited to, the N6 medium described by Chu et al. (1975) and MS media(Murashige and Skoog, 1962). It has been discovered that media such asMS that have a high ammonia/nitrate ratio are counterproductive to thegeneration of recipient cells in that they promote loss of morphogeniccapacity. N6 media, on the other hand, has a somewhat lowerammonia/nitrate ratio, and is contemplated to promote the generation ofrecipient cells by maintaining cells in a proembryonic state capable ofsustained divisions.

C. Maintenance

The method of maintenance of cell cultures may contribute to theirutility as sources of recipient cells for transformation. Manualselection of cells for transfer to fresh culture medium, frequency oftransfer to fresh culture medium, composition of culture medium, andenvironmental factors including, but not limited to, light quality andquantity and temperature are all important factors in maintaining callusand/or suspension cultures that are useful as sources of recipientcells. It is contemplated that alternating callus between differentculture conditions may be beneficial in enriching for recipient cellswithin a culture. For example, it is proposed that cells may be culturedin suspension culture, but transferred to solid medium at regularintervals. After a period of growth on solid medium cells can bemanually selected for return to liquid culture medium. It is proposedthat by repeating this sequence of transfers to fresh culture medium itis possible to enrich for recipient cells. It also is contemplated thatpassing cell cultures through a 1.9 mm sieve is useful in maintainingthe friability of a callus or suspension culture and may be beneficialin enriching for transformable cells.

D. Cryopreservation Methods

Cryopreservation is important because it allows one to maintain andpreserve a known transformable cell culture for future use, whileeliminating the cumulative detrimental effects associated with extendedculture periods.

Cell suspensions and callus were cryopreserved using modifications ofmethods previously reported (Finkle, 1985; Withers & King, 1979). Thecryopreservation protocol comprised adding a pre-cooled (0° C.)concentrated cryoprotectant mixture stepwise over a period of one to twohours to pre-cooled (0° C.) cells. The mixture was maintained at 0° C.throughout this period. The volume of added cryoprotectant was equal tothe initial volume of the cell suspension (1:1 addition), and the finalconcentration of cryoprotectant additives was 10% dimethyl sulfoxide,10% polyethylene glycol (6000 MW), 0.23 M proline and 0.23 M glucose.The mixture was allowed to equilibrate at 0° C. for 30 minutes, duringwhich time the cell suspension/cryoprotectant mixture was divided into1.5 ml aliquot (0.5 ml packed cell volume) in 2 ml polyethylenecryo-vials. The tubes were cooled at 0.5° C./minute to −8° C. and heldat this temperature for ice nucleation.

Once extracellular ice formation had been visually confirmed, the tubeswere cooled at 0.5° C./minute from −8° C. to −35° C. They were held atthis temperature for 45 minutes (to insure uniform freeze-induceddehydration throughout the cell clusters). At this point, the cells hadlost the majority of their osmotic volume (i.e., there is little freewater left in the cells), and they could be safely plunged into liquidnitrogen for storage. The paucity of free water remaining in the cellsin conjunction with the rapid cooling rates from −35° C. to −196° C.prevented large organized ice crystals from forming in the cells. Thecells are stored in liquid nitrogen, which effectively immobilizes thecells and slows metabolic processes to the point where long-term storageshould not be detrimental.

Thawing of the extracellular solution was accomplished by removing thecryo-tube from liquid nitrogen and swirling it in sterile 42° C. waterfor approximately 2 minutes. The tube was removed from the heatimmediately after the last ice crystals had melted to prevent heatingthe tissue. The cell suspension (still in the cryoprotectant mixture)was pipetted onto a filter, resting on a layer of BMS cells (the feederlayer that provided a nurse effect during recovery). The cryoprotectantsolution is removed by pipetting. Culture medium comprised a callusproliferation medium with increased osmotic strength. Dilution of thecryoprotectant occurred slowly as the solutes diffused away through thefilter and nutrients diffused upward to the recovering cells. Oncesubsequent growth of the thawed cells was noted, the growing tissue wastransferred to fresh culture medium. If initiation of a suspensionculture was desired, the cell clusters were transferred back into liquidsuspension medium as soon as sufficient cell mass had been regained(usually within 1 to 2 weeks). Alternatively, cells were cultured onsolid callus proliferation medium. After the culture was reestablishedin liquid (within 1 to 2 additional weeks), it was used fortransformation experiments. When desired, previously cryopreservedcultures may be frozen again for storage.

VI. Production and Characterization of Stably Transformed Plants

After effecting delivery of exogenous DNA to recipient cells, the nextsteps generally concern identifying the transformed cells for furtherculturing and plant regeneration. As mentioned herein, in order toimprove the ability to identify transformants, one may desire to employa selectable or screenable marker gene as, or in addition to, theexpressible gene of interest. In this case, one would then generallyassay the potentially transformed cell population by exposing the cellsto a selective agent or agents, or one would screen the cells for thedesired marker gene trait.

A. Selection

It is believed that DNA is introduced into only a small percentage oftarget cells in any one experiment. In order to provide an efficientsystem for identification of those cells receiving DNA and integratingit into their genomes one may employ a means for selecting those cellsthat are stably transformed. One exemplary embodiment of such a methodis to introduce into the host cell, a marker gene that confersresistance to some normally inhibitory agent, such as an antibiotic orherbicide. Examples of antibiotics that may be used include theaminoglycoside antibiotics neomycin, kanamycin and paromomycin, or theantibiotic hygromycin. Resistance to the aminoglycoside antibiotics isconferred by aminoglycoside phosphostransferase enzymes such as neomycinphosphotransferase II (NPT II) or NPT I, whereas resistance tohygromycin is conferred by hygromycin phosphotransferase.

Potentially transformed cells then are exposed to the selective agent.In the population of surviving cells will be those cells where,generally, the resistance-conferring gene has been integrated andexpressed at sufficient levels to permit cell survival. Cells may betested further to confirm stable integration of the exogenous DNA. Usingthe techniques disclosed herein, greater than 40% of bombarded embryosmay yield transformants.

One herbicide that constitutes a desirable selection agent is the broadspectrum herbicide bialaphos. Bialaphos is a tripeptide antibioticproduced by Streptomyces hygroscopicus and is composed ofphosphinothricin (PPT), an analogue of L-glutamic acid, and twoL-alanine residues. Upon removal of the L-alanine residues byintracellular peptidases, the PPT is released and is a potent inhibitorof glutamine synthetase (GS), a pivotal enzyme involved in ammoniaassimilation and nitrogen metabolism (Ogawa et al., 1973). SyntheticPPT, the active ingredient in the herbicide LIBERTY® also is effectiveas a selection agent. Inhibition of GS in plants by PPT causes the rapidaccumulation of ammonia and death of the plant cells.

The organism producing bialaphos and other species of the genusStreptomyces also synthesizes an enzyme phosphinothricin acetyltransferase (PAT) that is encoded by the bar gene in Streptomyceshygroscopicus and the pat gene in Streptomyces viridochromogenes. Theuse of the herbicide resistance gene encoding phosphinothricin acetyltransferase (PAT) is referred to in U.S. Pat. No. 5,276,268, wherein thegene is isolated from Streptomyces viridochromogenes. In the bacterialsource organism, this enzyme acetylates the free amino group of PPTpreventing auto-toxicity (Thompson et al., 1987). The bar gene has beencloned (Murakami et al., 1986; Thompson et al., 1987) and expressed intransgenic tobacco, tomato, potato (De Block et al., 1987) Brassica (DeBlock et al., 1989) and maize (U.S. Pat. No. 5,550,318). In previousreports, some transgenic plants that expressed the resistance gene werecompletely resistant to commercial formulations of PPT and bialaphos ingreenhouses.

Another example of a herbicide that is useful for selection oftransformed cell lines in the practice of the invention is the broadspectrum herbicide glyphosate. Glyphosate inhibits the action of theenzyme EPSPS, which is active in the aromatic amino acid biosyntheticpathway. Inhibition of this enzyme leads to starvation for the aminoacids phenylalanine, tyrosine, and tryptophan and secondary metabolitesderived thereof. U.S. Pat. No. 4,535,060 describes the isolation ofEPSPS mutations that confer glyphosate resistance on the Salmonellatyphimurium gene for EPSPS, aroA. The EPSPS gene was cloned from Zeamays and mutations similar to those found in a glyphosate resistant aroAgene were introduced in vitro. Mutant genes encoding glyphosateresistant EPSPS enzymes are described in, for example, PCT PublicationNo. WO 97/04103. The best characterized mutant EPSPS gene conferringglyphosate resistance comprises amino acid changes at residues 102 and106, although it is anticipated that other mutations will also be useful(PCT Publication No. WO97/04103). Furthermore, a naturally occurringglyphosate resistant EPSPS may be used, e.g., the CP4 gene isolated fromAgrobacterium encodes a glyphosate resistant EPSPS (U.S. Pat. No.5,627,061).

To use the bar-bialaphos or the EPSPS-glyphosate selective system,bombarded tissue is cultured for 0-28 days on nonselective medium andsubsequently transferred to medium containing from 1-3 mg/l bialaphos or1-3 mM glyphosate as appropriate. While ranges of 1-3 mg/l bialaphos or1-3 mM glyphosate will typically be preferred, it is proposed thatranges of 0.1-50 mg/l bialaphos or 0.1-50 mM glyphosate will findutility in the practice of the invention. Tissue can be placed on anyporous, inert, solid or semi-solid support for bombardment, includingbut not limited to filters and solid culture medium. Bialaphos andglyphosate are provided as examples of agents suitable for selection oftransformants, but the technique of this invention is not limited tothem.

It further is contemplated that the herbicide dalapon,2,2-dichloropropionic acid, may be useful for identification oftransformed cells. The enzyme 2,2-dichloropropionic acid dehalogenase(deh) inactivates the herbicidal activity of 2,2-dichloropropionic acidand therefore confers herbicidal resistance on cells or plantsexpressing a gene encoding the dehalogenase enzyme (Buchanan-Wollastonet al., 1992; U.S. Pat. No. 5,780,708).

Alternatively, a gene encoding anthranilate synthase, which confersresistance to certain amino acid analogs, e.g., 5-methyltryptophan or6-methyl anthranilate, may be useful as a selectable marker gene. Theuse of an anthranilate synthase gene as a selectable marker wasdescribed in U.S. Pat. No. 5,508,468 and PCT Publication No. WO97/26366.

An example of a screenable marker trait is the red pigment producedunder the control of the R-locus in maize. This pigment may be detectedby culturing cells on a solid support containing nutrient media capableof supporting growth at this stage and selecting cells from colonies(visible aggregates of cells) that are pigmented. These cells may becultured further, either in suspension or on solid media. The R-locus isuseful for selection of transformants from bombarded immature embryos.In a similar fashion, the introduction of the C1 and B genes will resultin pigmented cells and/or tissues.

The enzyme luciferase may be used as a screenable marker in the contextof the present invention. In the presence of the substrate luciferin,cells expressing luciferase emit light that can be detected onphotographic or x-ray film, in a luminometer (or liquid scintillationcounter), by devices that enhance night vision, or by a highly lightsensitive video camera, such as a photon counting camera. All of theseassays are nondestructive and transformed cells may be cultured furtherfollowing identification. The photon counting camera is especiallyvaluable as it allows one to identify specific cells or groups of cellsthat are expressing luciferase and manipulate those in real time.Another screenable marker that may be used in a similar fashion is thegene coding for green fluorescent protein.

It further is contemplated that combinations of screenable andselectable markers will be useful for identification of transformedcells. In some cell or tissue types a selection agent, such as bialaphosor glyphosate, may either not provide enough killing activity to clearlyrecognize transformed cells or may cause substantial nonselectiveinhibition of transformants and nontransformants alike, thus causing theselection technique to not be effective. It is proposed that selectionwith a growth inhibiting compound, such as bialaphos or glyphosate atconcentrations below those that cause 100% inhibition followed byscreening of growing tissue for expression of a screenable marker genesuch as luciferase would allow one to recover transformants from cell ortissue types that are not amenable to selection alone. It is proposedthat combinations of selection and screening may enable one to identifytransformants in a wider variety of cell and tissue types. This may beefficiently achieved using a gene fusion between a selectable markergene and a screenable marker gene, for example, between an NPTII geneand a GFP gene.

B. Regeneration and Seed Production

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. In an exemplary embodiment, MS andN6 media may be modified (see Table 7) by including further substancessuch as growth regulators. A preferred growth regulator for suchpurposes is dicamba or 2,4-D. However, other growth regulators may beemployed, including NAA, NAA+2,4-D or perhaps even picloram. Mediaimprovement in these and like ways has been found to facilitate thegrowth of cells at specific developmental stages. Tissue may bemaintained on a basic media with growth regulators until sufficienttissue is available to begin plant regeneration efforts, or followingrepeated rounds of manual selection, until the morphology of the tissueis suitable for regeneration, at least 2 wk, then transferred to mediaconducive to maturation of embryoids. Cultures are transferred every 2wk on this medium. Shoot development will signal the time to transfer tomedium lacking growth regulators.

The transformed cells, identified by selection or screening and culturedin an appropriate medium that supports regeneration, will then beallowed to mature into plants. Developing plantlets are transferred tosoiless plant growth mix, and hardened off, e.g., in an environmentallycontrolled chamber at about 85% relative humidity, 600 ppm CO₂, and25-250 microeinsteins m⁻²s⁻¹ of light, prior to transfer to a greenhouseor growth chamber for maturation. Plants are preferably matured eitherin a growth chamber or greenhouse. Plants are regenerated from about 6wk to 10 months after a transformant is identified, depending on theinitial tissue. During regeneration, cells are grown on solid media intissue culture vessels. Illustrative embodiments of such vessels arepetri dishes and Plant Cons. Regenerating plants are preferably grown atabout 19 to 28° C. After the regenerating plants have reached the stageof shoot and root development, they may be transferred to a greenhousefor further growth and testing.

Note, however, that seeds on transformed plants may occasionally requireembryo rescue due to cessation of seed development and prematuresenescence of plants. To rescue developing embryos, they are excisedfrom surface-disinfected seeds 10-20 days post-pollination and cultured.An embodiment of media used for culture at this stage comprises MSsalts, 2% sucrose, and 5.5 g/l agarose. In embryo rescue, large embryos(defined as greater than 3 mm in length) are germinated directly on anappropriate media. Embryos smaller than that may be cultured for 1 wk onmedia containing the above ingredients along with 10⁻⁵M abscisic acidand then transferred to growth regulator-free medium for germination.

Progeny may be recovered from transformed plants and tested forexpression of the exogenous expressible gene by localized application ofan appropriate substrate to plant parts such as leaves. In the case ofbar transformed plants, it was found that transformed parental plants(R_(O)) and their progeny of any generation tested exhibited nobialaphos-related necrosis after localized application of the herbicideBasta to leaves, if there was functional PAT activity in the plants asassessed by an in vitro enzymatic assay. All PAT positive progeny testedcontained bar, confirming that the presence of the enzyme and theresistance to bialaphos were associated with the transmission throughthe germline of the marker gene.

C. Characterization

To confirm the presence of the isolated and purified DNA segment(s) or“transgene(s)” in the regenerating plants, a variety of assays may beperformed. Such assays include, for example, “molecular biological”assays well known to those of skill in the art, such as Southern andNorthern blotting, RT-PCR and PCR; “biochemical” assays, such asdetecting the presence of a protein product, e.g., by immunologicalmeans (ELISAs and Western blots) or by enzymatic function; plant partassays, such as leaf or root assays; and also, by analyzing thephenotype of the whole regenerated plant.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA may only be expressed in particular cells ortissue types and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR techniques may also be used for detection andquantitation of RNA produced from introduced isolated and purified DNAsegments. In this application of PCR it is first necessary to reversetranscribe RNA into DNA, using enzymes such as reverse transcriptase,and then through the use of conventional PCR techniques amplify the DNA.In most instances PCR techniques, while useful, will not demonstrateintegrity of the RNA product. Further information about the nature ofthe RNA product may be obtained by Northern blotting. This techniquewill demonstrate the presence of an RNA species and give informationabout the integrity of that RNA. The presence or absence of an RNAspecies can also be determined using dot or slot blot Northernhybridizations. These techniques are modifications of Northern blottingand will only demonstrate the presence or absence of an RNA species.

While Southern blotting and PCR may be used to detect the isolated andpurified DNA segment in question, they do not provide information as towhether the isolated and purified DNA segment is being expressed.Expression may be evaluated by specifically identifying the proteinproducts of the introduced isolated and purified DNA sequences orevaluating the phenotypic changes brought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focussing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as Western blotting in which antibodies are used locateindividual gene products that have been separated by electrophoretictechniques. Additional techniques may be employed to absolutely confirmthe identity of the product of interest such as evaluation by amino acidsequencing following purification. Although these are among the mostcommonly employed, other procedures may be additionally used.

Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of isolated andpurified DNA segments encoding storage proteins that change amino acidcomposition and may be detected by amino acid analysis.

1. DNA Integration, RNA Expression and Inheritance

Genomic DNA may be isolated from callus cell lines or any plant parts todetermine the presence of the isolated and purified DNA segment throughthe use of techniques well known to those skilled in the art. Note thatintact sequences will not always be present, presumably due torearrangement or deletion of sequences in the cell.

The presence of DNA elements introduced through the methods of thisinvention may be determined by polymerase chain reaction (PCR). Usingthis technique discreet fragments of DNA are amplified and detected bygel electrophoresis. This type of analysis permits one to determinewhether an isolated and purified DNA segment is present in a stabletransformant, but does not prove integration of the introduced isolatedand purified DNA segment into the host cell genome. In addition, it isnot possible using PCR techniques to determine whether transformantshave exogenous genes introduced into different sites in the genome,i.e., whether transformants are of independent origin. It iscontemplated that using PCR techniques it would be possible to clonefragments of the host genomic DNA adjacent to an introduced isolated andpurified DNA segment.

Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique specific DNAsequences that were introduced into the host genome and flanking hostDNA sequences can be identified. Hence the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition it is possible through Southernhybridization to demonstrate the presence of introduced isolated andpurified DNA segments in high molecular weight DNA, i.e., confirm thatthe introduced isolated and purified DNA segment has been integratedinto the host cell genome. The technique of Southern hybridizationprovides information that is obtained using PCR, e.g., the presence ofan isolated and purified DNA segment, but also demonstrates integrationinto the genome and characterizes each individual transformant.

It is contemplated that using the techniques of dot or slot blothybridization that are modifications of Southern hybridizationtechniques one could obtain the same information that is derived fromPCR, e.g., the presence of an isolated and purified DNA segment.However, it is well known in the art that dot or slot blot hybridizationmay produce misleading results, as probe may be non-specifically boundby high molecular weight DNA.

Both PCR and Southern hybridization techniques can be used todemonstrate transmission of an isolated and purified DNA segment toprogeny. In most instances the characteristic Southern hybridizationpattern for a given transformant will segregate in progeny as one ormore Mendelian genes (Spencer et al., 1992; Laursen et al., 1994)indicating stable inheritance of the gene. For example, in one study, of28 progeny (R₁) plants tested, 50% (N=14) contained bar, confirmingtransmission through the germline of the marker gene. The nonchimericnature of the callus and the parental transformants (R₀) was suggestedby germline transmission and the identical Southern blot hybridizationpatterns and intensities of the transforming DNA in callus, R₀ plantsand R₁ progeny that segregated for the transformed gene.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA may only be expressed in particular cells ortissue types and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR techniques may also be used for detection andquantitation of RNA produced from introduced isolated and purified DNAsegments. In this application of PCR it is first necessary to reversetranscribe RNA into DNA, using enzymes such as reverse transcriptase,and then through the use of conventional PCR techniques amplify the DNA.In most instances PCR techniques, while useful, will not demonstrateintegrity of the RNA product. Further information about the nature ofthe RNA product may be obtained by Northern blotting. This techniquewill demonstrate the presence of an RNA species and give informationabout the integrity of that RNA. The presence or absence of an RNAspecies can also be determined using dot or slot blot Northernhybridizations. These techniques are modifications of Northern blottingand will only demonstrate the presence or absence of an RNA species.

2. Gene Expression

While Southern blotting and PCR may be used to detect the isolated andpurified DNA segment in question, they do not provide information as towhether the isolated and purified DNA segment is being expressed.Expression may be evaluated by specifically identifying the proteinproducts of the introduced isolated and purified DNA segments orevaluating the phenotypic changes brought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focussing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

Assay procedures may also be used to identify the expression of proteinsby their functionality, especially the ability of enzymes to catalyzespecific chemical reactions involving specific substrates and products.These reactions may be followed by providing and quantifying the loss ofsubstrates or the generation of products of the reactions by physical orchemical procedures. Examples are as varied as the enzyme to be analyzedand may include assays for PAT enzymatic activity by followingproduction of radiolabeled acetylated phosphinothricin fromphosphinothricin and ¹⁴C-acetyl CoA or for anthranilate synthaseactivity by following loss of fluorescence of anthranilate, to name two.

Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of isolated andpurified DNA segments encoding enzymes or storage proteins which changeamino acid composition and may be detected by amino acid analysis, or byenzymes that change starch quantity that may be analyzed by nearinfrared reflectance spectrometry. Morphological changes may includegreater stature or thicker stalks. Most often changes in response ofplants or plant parts to imposed treatments are evaluated undercarefully controlled conditions termed bioassays.

D. Establishment of the Introduced DNA in Other Plant Varieties

Fertile, transgenic plants may then be used in a conventional breedingprogram in order to incorporate the isolated and purified DNA segmentinto the desired lines or varieties. Methods and references forconvergent improvement of are given by Hallauer et al. (1988),incorporated herein by reference. Among the approaches that conventionalbreeding programs employ is a conversion process (backcrossing).Briefly, conversion is performed by crossing the initial transgenicfertile plant to elite inbred lines (which may or may not be transgenicto yield an F₁ hybrid plant). The progeny from this cross will segregatesuch that some of the plants will carry the isolated and purified DNAsegment whereas some will not. The plants that do carry the isolated andpurified DNA segment are then crossed again to the elite inbred linesresulting in progeny that segregate once more. This backcrossing processis repeated until the original elite inbred has been converted to a linecontaining the isolated and purified DNA segment, yet possessing allimportant attributes originally found in the parent. Generally, thiswill require about 6-8 generations. Then the resultant F_(n) hybrid isselfed at least 5-7 times to yield an inbred line. A separatebackcrossing program will be generally used for every elite line that isto be converted to a genetically engineered elite line.

Generally, the commercial value of the transformed plants producedherein will be greatest if the isolated and purified DNA segment can beincorporated into many different hybrid combinations. A farmer typicallygrows several hybrids based on differences in maturity, standability,and other agronomic traits. Also, the farmer must select a hybrid basedupon his or her geographic location since hybrids adapted to one regionare generally not adapted to another because of differences in suchtraits as maturity, disease, drought and insect resistance. As such, itis necessary to incorporate the gene into a large number of parentallines so that many hybrid combinations can be produced containing theisolated and purified DNA segment.

Plant breeding and the techniques and skills required to transfer genesfrom one line or variety to another are well known to those skilled inthe art. Thus, introducing an isolated and purified DNA segment,preferably in the form of recombinant DNA, into any other line orvariety can be accomplished by these breeding procedures.

E. Alteration of Transgene Insertions

At anytime during the process of incorporation of a transgene into othervarieties of the plant species, alterations in the transgene insertionmay be induced and selected. Preferably, alterations are induced earlyin the process of incorporating the transgene insertion into othervarieties, so as to minimize the number of further variety conversions,e.g., backcross conversions, that must be made after the alteredtransgene insertion is selected. The use of non-homologous recombinationto alter a transgene insertion requires the presence of a directlyrepeated DNA sequence within the transgene insertion. Directly repeatedsequences may be present on a plasmid vector when introduced in a plant.For example, plasmid pMON19344 (FIG. 6) comprises a cryIA(b) geneflanked by directly repeated P-e35S and hsp70 intron sequences.Furthermore, the nptII gene in pMON19344 is flanked by directly repeatednopaline synthase (NOS) 3′ sequences. Similarly, a single transgene on aplasmid vector may be flanked by directly repeated sequences.Integration of plasmid vectors such as pMON19344 as a linear transgeneinsertion leads to both the cryIA(b) and nptII genes being flanked bydirectly repeated sequences.

Alternatively, directly repeated DNA sequences may be generated in thetransgene insertion by rearrangements, duplications and tandemintegrations of DNA sequences in the transgene insertion. Therefore,although directly repeated DNA sequences are not present on the plasmidvectors that are introduced into the plants, direct repeats are producedduring the DNA integration process. Regardless of the process used, theresult is a transgene insertion comprising directly repeated DNAsequences.

As the process of the present invention utilizes a plant's naturallyoccurring DNA recombination processes, the transgene insertion must passthrough at least one cycle of meiosis in a cell in order for alterationsto occur. Although somatic recombination is known in plants (Evans andPaddock, 1979), only those recombination events that result inalterations in the transgene insertion in germ line cells will transmitthe alteration to progeny. Furthermore, in order for meioticrecombination to occur a transgene insertion must be present in at leasttwo separate locations in the host genome, preferably on sisterchromosome pairs. Although duplicate transgene insertions may be presentat locations that do not lie on sister chromosomes, recombinationbetween such transgene insertions may give rise to chromosomalrearrangements, thereby producing undesirable phenotypic results in thecell or plant.

In the normal course of plant transformation a transgene insertionoccurs at a single chromosomal locus. Therefore, the transformed celland directly derived transformed plant contain a single copy of thetransgene insertion, i.e., the cell and plant are hemizygous. In such ahemizygous plant, there is no additional copy of the transgene insertionto pair with and undergo meiotic recombination. Therefore, alteration ofthe transgene insertion cannot occur in a hemizygous plant. It is,therefore, necessary to produce a plant comprising the transgeneinsertion on both of a pair of sister chromosomes, i.e., a plant that ishomozygous for the transgene insertion. Such a homozygous transgeneinsertion plant may be produced by conventional breeding, i.e., selfpollination and identification of a non-segregating transgenic progenypopulation. Meiotic recombination, e.g., the desired non-homologousrecombination occurs during meiosis in the homozygous plant andnon-homologous recombinants are present in the progeny population.Non-homologous recombinants may be identified in progeny producedthrough self-fertilization or outcrossing to a plant lacking thetransgene insertion. Preferably, plants comprising homozygous transgenicinsertions are crossed to non-transgenic plants in order to simplifyidentification of recombinants.

During the process of meiotic recombination, both homologous andnon-homologous recombinants will be produced. It is anticipated thathomologous recombinants will demonstrate gene expression similar to theparent plant. Selection of progeny plants comprising altered transgeneinsertions produced through non-homologous recombination is based onidentification of progeny plants with altered transgene expression,preferably loss of transgene expression. Altered expression may bedetected by a phenotypic assay, e.g., herbicide resistance or insectresistance, or direct assays for enzyme activity or presence of thetransgene encoded protein. The presence of an altered transgeneinsertion is likely in progeny plants in which transgene expressiondiffers from expression in the parent transgenic plant. Alterations inthe transgene insertion may be confirmed by PCR or Southern blotanalysis.

F. Uses of Transgenic Plants

The transgenic plants produced herein are expected to be useful for avariety of commercial and research purposes. Transgenic plants can becreated for use in traditional agriculture to possess traits beneficialto the grower (e.g., agronomic traits such as resistance to waterdeficit, pest resistance, herbicide resistance or increased yield),beneficial to the consumer of the grain harvested from the plant (e.g.,improved nutritive content in human food or animal feed), or beneficialto the food processor (e.g., improved processing traits). In such uses,the plants are generally grown for the use of their grain in human oranimal foods. However, other parts of the plants, including stalks,husks, vegetative parts, and the like, may also have utility, includinguse as part of animal silage or for ornamental purposes. Often, chemicalconstituents (e.g., oils or starches) of and other crops are extractedfor foods or industrial use and transgenic plants may be created thathave enhanced or modified levels of such components.

Transgenic plants may also find use in the commercial manufacture ofproteins or other molecules, where the molecule of interest is extractedor purified from plant parts, seeds, and the like. Cells or tissue fromthe plants may also be cultured, grown in vitro, or fermented tomanufacture such molecules.

The transgenic plants may also be used in commercial breeding programs,or may be crossed or bred to plants of related crop species.Improvements encoded by the isolated and purified DNA segment may betransferred, e.g., from cells to cells of other species, e.g., byprotoplast fusion.

The transgenic plants may have many uses in research or breeding,including creation of new mutant plants through insertional mutagenesis,in order to identify beneficial mutants that might later be created bytraditional mutation and selection. An example would be the introductionof a recombinant DNA sequence encoding a transposable element that maybe used for generating genetic variation. The methods of the inventionmay also be used to create plants having unique “signature sequences” orother marker sequences that can be used to identify proprietary lines orvarieties.

The following examples are illustrative of the present invention.

EXAMPLE 1 Deletion of the Bar Gene from the Transgenic Event DBT418

Non-reciprocal homologous recombination-mediated transgene deletion is aprocess whereby the structure of a transgene insert can be altered (seeFIG. 1). The process is dependent on the presence of direct repeats ofDNA sequences in the transgene insertion. Direct repeats may be presentin the transgene used for transformation, or they may arise throughmulti-element integration at the site of transgene insertion. The directrepeats might be, for example, incomplete parts of a transgene that,upon recombination, produce a complete transgene conferring anidentifiable phenotype.

Line DBT418 was produced by microprojectile bombardment of embryogeniccells with plasmid vectors pDPG354 (FIG. 5), pDPG165 (FIG. 6) andpDPG320 (FIG. 8). The structure of the transgene insert in the lineDBT418 is diagramed in FIG. 2 and described in detail in U.S.D.A.Petition 9629101p for deregulation. The insert has one functional copyof a bar gene conferring resistance to the herbicide phosphinothricin.Flanking the bar gene on both sides are directly repeated DNA sequencesthat include cloning vector DNA and Bt toxin encoding DNA sequences. Inaddition to these direct repeats, there are additional shorter regionsof direct homology that also may serve as target sequences fornon-reciprocal recombination mediated deletion of transgene DNA withinthe insert.

In order to identify individuals that have undergone non-reciprocalrecombination mediated transgene deletion, an assay was carried out toscreen for plants that showed a loss of the phosphinothricin resistancephenotype. Southern blot analysis was used to characterize the copynumber of transgene elements present in the phosphinothricin sensitiveindividuals.

Hemizygous DBT418 plants were selfed, and progeny identified that werehomozygous for the DBT418 insertion event. These homozygous plants wereoutcrossed to non-transgenic plants to generate a population ofhemizygous seed. Approximately 1,000 seed of a finished inbred,hemizygous for the DBT418 insert, were planted and assayed forphosphinothricin resistance using a nondestructive herbicide leafpainting assay (U.S. Pat. No. 5,489,520). Individuals displaying anecrotic response in the treated area were assayed again by the leafpainting assay for confirmation of the phosphinothricin sensitivephenotype. Five individuals were found to be sensitive tophosphinothricin.

Genomic DNA was isolated and analyzed by Southern blot analysis. Theblot was hybridized with probes for the Bt, bar and amp genes. Resultsof this analysis are shown in Table 8.

TABLE 8 Summary of DBT418 Recombinants Displaying PhosphinothricinSensitivity Phenotypes and Genotypes # full- # full- # partial # full-length Individual Pheno- length bar bar gene length Bt Amp Row Planttype^(a) gene copies copies gene copies copies 03 09 S 0 1 1 1 08 17 S 01 $3 >5 09 07 S 0 1 2 2 11 18 S 0 1 2 2 15 11 S 0 1 2 2 Normal R 1 1 2 3DBT418 ^(a)PPT resistant (R) or Sensitive (S)

All five phosphinothricin-sensitive individuals lacked the full lengthbar gene present in phosphinothricin-resistant DBT418. The data alsoshowed that each phosphinothricin-sensitive plant still containedtransgene DNA corresponding to the partial bar gene copy, the Bt geneand the amp gene. The copy number of these transgenes varied among thephosphinothricin sensitive individuals. The data shows three classes ofvariants. Plant 03-09 lacked the full length bar gene copy, but retaineda partial bar gene copy, one copy of the Bt gene and one copy of the ampgene. Plants 09-07, 11-18, and 15-11 represent a second class ofvariants that lacked the full length bar gene, but retained a partialbar gene, two copies of the Bt gene, and two copies of the amp gene.Finally, a third class was observed where the full length bar gene copywas absent, but a partial bar gene copy was retained, and where the copynumber of the Bt gene and amp gene were increased compared to DBT418.

A model for the non-reciprocal recombination-mediated deletion leadingto the generation of DBT418 variants 09-07 and 03-09 is presented inFIG. 3. The involvement of interchromosomal non-reciprocal recombinationin this process is supported by the fact that individual DBT418 variantswere identified that contained an increase in transgene copy number aswould be predicted from the model outlined in FIG. 1.

EXAMPLE 2 Deletion of nptII or cryIA(b) Gene from the Transgenic Events“MON849” and “MON850”

Transformation events (MON849) and (MON 850) were produced bymicroprojectile bombardment of cells with plasmid vector using pMON19344(FIG. 6). The structure of the MON849 transgene insert is diagramed inFIG. 4. The insert has one copy of an nptII gene conferring resistanceto kanamycin and one copy of a cryIA(b) Bt gene conferring resistance tocertain insect pests. Both the nptII and cryIA(b) coding regions areflanked on the 5′ ends by identical 35S promoters and hsp70 introns.Both the nptII and cryIA(b) coding regions are flanked on the 3′ ends byidentical nos terminators. Recombination events between the 35S promoterand hsp7o intron regions of the cryIA b) gene and the 35S promoter andhsp70 intron regions of the nptII gene result in the loss of thecryIA(b) gene (FIG. 4). Recombination events between the nos terminatorregion of the cryIA(b) gene and the nos terminator region of the nptIIgene result in the loss of the nptII gene (FIG. 4). The latterrecombination event is useful in that (I) the resultant plant would begenetically more stable, as loss of the cryIA(b) gene would not occurduring seed increase, (ii) the resultant plant would be phenotypicallymore stable, as there would be no repeated genetic elements within theinsert, and (iii) the ancillary DNA sequence encoding nptII that doesnot contribute to the designed insect resistance phenotype is deleted.

Plant material was prepared by self pollinating plants hemizygous forthe transgene insert, identifying individuals homozygous for thetransgene insert in the subsequent generation, and crossing thehomozygous individuals to nontransgenic plants. The resulting populationwas hemizygous for the transgene insert.

To identify non-reciprocal recombinants within this MON849 progenypopulation, transgene expression assays were carried out onapproximately 1,000 individuals and 7 individuals that differed inphenotype from the parent were identified (Table 9) (frequency of 0.4%).PCR analysis carried out for the cryIA(b) and nptII genes showed thatthe lack of a transgene phenotype correlated with the absence of theparticular transgene. Plant 20-102-A (plant numbers refer torange-row-stake number, as listed in Table 9) appears to be arecombinant that has lost the nptII gene. Plant 20-103-3 lacks bothtransgenes and may be the result of pollen contamination. Five MON849progeny plants show an apparent recombination in which the cryIA(b) genewas lost and the nptII gene retained. A similar transgene stabilityassay was also carried out on approximately 1,000 individuals derivedfrom a parent plant that was homozygous for the MON850 event and about0.7% of the individuals differed from the parent. One MON849 progenyplant and one MON850 progeny plant showed an apparent recombination inwhich the nptII gene was lost and the cryIA(b) gene retained. Therecombinant individuals lacking the nptII gene were crossed with avariety of inbreds.

TABLE 9 Genetic Analysis (PCR) of WHITMAN and GENESIS Plants DisplayingOff-type Phenotypes Phenotypes Genotypes (PCR) Event Range Row Stake #Cry1A(b) NPTII Cry1A(b) NPTII MON850 19 126  8¹ o o o o MON850 18 125 7¹ o o o o MON850 18 129 B + o + o MON850 19 125 C + o + + MON849 20102 A + o + o MON849 18 113 6 o + o + MON849 20 105 5 o + o + MON849 19105 4 o + o + MON849 20 103 3 o o o o MON849 20 99 2 o + o + MON849 1999 1 o + o 0 ¹These plants were small in stature, consistent with theseindividuals being nontransgenic inbred.

Southern blot analyses of the recombinant MON849 individuals werecarried out in order to confirm that gene deletion was mediated bynon-reciprocal recombination. As shown in Table 10, bothnptII+/cryIA(b)− and nptII−/cryIA(b)+ individuals displayed a pattern ofhybridizing bands that are indicative of non-reciprocal recombinationmediated transgene deletion.

TABLE 10 Southern hybridization band sizes for MON849 F1 derivativesProbe A = Phenotype Cry1A(b) Probe B = nptII Cry1A(b) % in F₁ EcoRI NcoIEcoRI (E) − Kan^(R) ELISA progeny¹ (E) (N) NcoI (N) XbaI (X) + + 99.3%10.0 6.1 2.6 5.2 + o 0.6% ? ? 7.3 5.2 o + 0.1% 10.0 5.9 ? ? ¹n = 1000

Quantitative ELISA analysis of a nptII−/cryIA(b)+ individual derivedfrom both MON849 and MON850 events indicated that deletion of the nptIIgene did not significantly compromise the expression of the cryIA(b)gene as shown in Table 11.

TABLE 11 Quantitative ELISA on MON849 and MON850 F1 derivativesPhenotype Cry1A(b) Protein (μg/g dry wt.) Kan^(R) Cry1A(b) MON849MON850 + + 18.48 11.22 o + 11.59 17.36

Finally, in looking at the relationship between the repeated sequencesflanking the deleted gene and the frequency of recombination, a directcorrelation was observed between the length of the direct repeatsequences flanking the deleted gene and the observed frequency ofnon-reciprocal recombination mediated transgene deletion (Table 12). Theobserved gene deletion frequency is estimated at about 0.1% per 287 bpof homologous direct repeat sequence ±19 bp (S.E).

TABLE 12 Correlation Between Flanking Direct Repeat Length and Frequencyof Intervening Gene Deletion Deleted Direct Repeat % Deletion Event GeneRepeats Length Recombinants Whitman nptII nos 0.3 kbp 0.1% WhitmanCry1A(b) e35S-hsp70 1.5 kbp 0.6% DBT418 bar pDPG354 6.2 kbp 2.0%

In conclusion, non-reciprocal recombination can be used to removeunwanted transgenic DNA sequences from genetically transformed plants.Target trait gene expression was not compromised by the deletion of alinked marker gene. Moreover, the observed recombination frequencyappears to be directly proportional to the length of the repeats withinthe region being targeted for gene deletion. Thus, transformation can bedesigned to facilitate subsequent gene deletion, such as in pMON19344.

All publications, patents and patent applications cited above areincorporated by reference herein, as though fully set forth. Theinvention has been described with reference to various specific andpreferred embodiments and will be further described by reference to thefollowing detailed examples. It is understood, however, that there aremany extensions, variations, and modifications on the basic theme of thepresent invention beyond that shown in the examples and description,which are within the spirit and scope of the present invention.

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What is claimed is:
 1. A method of preparing a fertile transgenic cerealplant having an altered transgene insertion, comprising: a) obtaining afirst fertile transgenic cereal plant homozygous for a transgeneinsertion DNA sequence, wherein the transgene insertion DNA sequencecomprises a pre-selected DNA sequence flanked by directly repeated DNAsequences, wherein said directly repeated sequences are not recognizedby a site-specific recombinase enzyme; b) obtaining a plurality ofprogeny of any generation of the first fertile transgenic cereal plant;and c) selecting a progeny fertile transgenic cereal plant wherein atleast a portion of the transgene insertion is altered as compared to thefirst fertile transgenic cereal plant.
 2. The method of claim 1 whereinthe pre-selected DNA sequence comprises a selectable marker gene or areporter gene.
 3. The method of claim 1 wherein the pre-selected DNAsequence comprises a bar, nptII, or cryIA(b) gene.
 4. The method ofclaim 1 wherein the plurality of progeny plants are obtained byself-pollination.
 5. The method of claim 1 wherein the plurality ofprogeny plants are obtained by outcrossing to produce hybrid progeny. 6.The method of claim 1 wherein the plurality of progeny plants areobtained by inbreeding to produce inbred plants.
 7. The method of claim1 wherein the cereal plant is a maize, barley, wheat, rye or rice plant.8. The method of claim 7 wherein the plant is a maize plant.
 9. Themethod of claim 1 wherein at least a portion of the transgene insertionis altered in that it has been deleted, amplified, or rearranged.